Can DNA rings, i.e. plasmids, form as Möbius strips?

Can DNA rings, i.e. plasmids, form as Möbius strips?

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I know that plasmids can be present in a coiled form, which keeps the DNA strands together when they degenerate, by forming catenases.

I was wondering, however, whether it has been documented to naturally happen or if anybody has ever successfully created a Möbius-strip plasmid. Technically, such a structure could readily form from a single circular strand of DNA, but the literature, as far as I have searched, has remained silent.

Knots and nonorientable surfaces in chiral nematics

Knots and knotted fields enrich physical phenomena ranging from DNA and molecular chemistry to the vortices of fluid flows and textures of ordered media. Liquid crystals provide an ideal setting for exploring such topological phenomena through control of their characteristic defects. The use of colloids in generating defects and knotted configurations in liquid crystals has been demonstrated for spherical and toroidal particles and shows promise for the development of novel photonic devices. Extending this existing work, we describe the full topological implications of colloids representing nonorientable surfaces and use it to construct torus knots and links of type (p,2) around multiply twisted Möbius strips.

Controlling and designing complex 3D textures in ordered media is central to the development of advanced materials, photonic crystals, tunable devices or sensors, and metamaterials (1 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ –10), as well as to furthering our basic understanding of mesophases (11 ⇓ –13). Topological concepts, in particular, have come to play an increasingly significant role in characterizing materials across a diverse range of topics from helicity in fluid flows (14, 15) and transitions in soap films (16) to molecular chemistry (17), knots in DNA (18), defects in ordered media (19, 20), quantum computation (21, 22), and topological insulators (23). Topological properties are robust, because they are protected against all continuous deformations, and yet flexible for the same reason, allowing for tunability without loss of functionality.

Some of the most intricate and interesting textures in ordered media involve knots. Originating with Lord Kelvin’s celebrated “vortex atom” theory (24), the idea of encoding knotted structures in continuous fields has continued in magnetohydrodynamics (25), fluid dynamics (15), high-energy physics (26 ⇓ –28), and electromagnetic fields (29, 30), and has seen recent experimental realizations in optics (31), liquid crystals (32), and fluid vortices (33). Tying knots in a continuous field involves a much greater level of complexity than in a necktie, or rope, or even a polymer or strand of DNA. In a field, the knot is surrounded by material that has to be precisely configured so as to be compatible with the knotted curve. However, this complexity brings its own benefits, for the full richness of the mathematical theory of knots is naturally expressed in terms of the properties of the knot complement: everything that is not the knot. In this sense, knotted fields are ideally suited to directly incorporate and experimentally realize the full scope of modern knot theory.

Liquid crystals are orientationally ordered mesophases, whose unique blend of soft elasticity, optical activity, and fluid nature offers a fertile setting for the development of novel metamaterials and the study of low-dimensional topology in ordered media. Much of the current focus centers on colloidal systems––colloidal particles dispersed in a liquid crystal host––which have a dual character. On the one hand, the liquid crystal mediates long-range elastic interactions between colloids, furnishing the mechanism for formation of colloidal structures and metamaterials (1 ⇓ –3, 10). On the other hand, the colloids, through anchoring conditions imposed by their surfaces, generate defects in the liquid crystal and so serve to induce and manipulate its topological properties. For instance, multiple colloids exhibit a variety of entangled defect configurations (34, 35), equally interesting states without defects (36), and can even be manipulated so as to form arbitrary knots and links (32, 37). More recently, a significant advance has seen the fabrication of colloids with different topology (38)––tori up to genus five––verifying experimentally the relation between particle topology and accompanying defect charge, and advancing a program to obtain topological control of materials through topological design. Although the phenomena displayed by these systems are indeed rich, as surfaces these colloids (spheres, tori, etc.) all represent closed, orientable surfaces.

In this article we extend these ideas to provide a complete topological characterization of all compact colloidal surfaces in a liquid crystal host. Nonorientable surfaces fully exploit the nonorientable nature of liquid crystalline order (39). On all nonorientable surfaces there are, by necessity, closed paths around which the surface normal reverses its orientation. For surfaces with normal anchoring this imprints a corresponding reversal in the director field, the telltale signature of a disclination line. In this way nonorientable surfaces enforce the creation of topologically protected dislcination lines. By varying the embeddings of the surface we exploit this topology to create metastable disclination loops in the shape of torus knots and links, for any p, around multiply twisted Möbius bands. Through this combination of geometry and topology we elucidate a natural setting for the creation and control of complex knotted fields and the integration of mathematical knot theory into experimental science.


Bacterial conjugation is the process by which DNA is transferred unidirectionally from a donor cell to a recipient cell. It plays a crucial role in horizontal gene transfer, the major means by which bacteria evolve and adapt to their environment, and also a process of immense biomedical importance since conjugation is the main vector of propagation of antibiotics resistance genes. It was first described by Lederberg and Tatum in the 1940s 1 . Its discovery signalled the dawn of molecular biology once it was demonstrated that the transfer of genetic information was unidirectional and that the entire genome of Escherichia coli could be passed from one cell to another starting at a defined site 2 . Indeed, landmark discoveries followed: the mapping of the E. coli genome (mapped in “minutes”, i.e. the time taken by a particular gene to be transferred from donor to recipient, with time 0 being the mating start—when donor and recipient cells were put in the presence of each other) or the discovery of gene structure and regulation (please refer to the fascinating account of this research in the Nobel lectures by the founding fathers of the field of molecular biology, Francois Jacob, Andre Lwoff and Jacques Monod in 1965 3 ).

The various machineries utilized during conjugation to execute DNA transfer are usually encoded by conjugative plasmids or other genetic mobile elements such as integrated conjugative elements (ICE). Plasmids are ubiquitous in bacteria and are defined as a collection of genetic modules organized into a stable, usually circular, self-replicating replicon, which does not usually contain genes essential for cell functions (reviewed in ref. 4 ). Several of these modules contain genes encoding proteins that assemble into large complexes mediating most commonly the plasmid's own transfer to a recipient bacterial cell, but also intriguingly (but rarely) to a eukaryotic cell such as yeast, plant or human cells 5-7 . Interestingly, these modules are evolutionary related to clusters found in genomic islands of a restricted number of bacterial pathogens such as Helicobacter pylori, Bordetella pertussis or Legionella pneumophila where they play essential roles in pathogenicity by injecting protein effectors into eukaryotic hosts 8 (Fig 1).

Figure 1. The various processes in which T4S systems are involved

Conjugation in Gram-negative bacteria is mediated by three large complexes: a DNA-processing machinery called “the relaxosome” a membrane-embedded transport machinery termed “type IV secretion (T4S) system” and a pilus 9 .

Conjugation starts with the assembly of the relaxosome to a particular site on the plasmid DNA called the “origin of transfer” or OriT. The relaxosome includes one key protein called the “relaxase” and a number of accessory proteins. The relaxase plays essential roles: (i) it catalyses a nicking reaction on a single strand of OriT DNA at the so-called nic site and covalently reacts to the 5′-phosphate generated by the nicking reaction and (ii) it binds to the T4S system through interactions with one of the constituents of the transport machinery, the coupling protein (reviewed in ref. 10 ).

The T4S system is one of six secretion systems embedded in both membranes of Gram-negative bacteria 11 . Minimally, they are composed of 12 proteins termed “VirB1-11 and VirD4” (to use the naming nomenclature derived from the Agrobacterium tumefaciens T4S system) 12 . Three components, VirB7, VirB9 and VirB10, form the so-called outer-membrane core complex (OMCC), absent in Gram-positive T4S systems where there is no OM 13 . The OMCC connect to an inner-membrane complex (IMC) composed of VirD4, VirB4, VirB3, VirB6, VirB8 and part of VirB10. OMCC and IMC are connected through a stalk of unknown composition, perhaps made of VirB2 and VirB5 14 or VirB10 14-16 . At least two ATPases (VirB4 and VirD4), or sometimes three (VirB4, VirD4 and VirB11), power the system.

Finally, the conjugative pilus of Gram-negative bacteria is an essential element in conjugation. For decades, it was the only feature in conjugating cells that could be observed or purified 17 . It is made of a major component, VirB2, and a minor one, VirB5. VirB2 assembles into a large helical filament with perhaps VirB5 at its tip 18 . Pili have been hypothesized to either serve as attachment devices mediating recognition of and attachment to recipient cells or serve as a conduit for relaxase/ssDNA transport, or both. Some conjugative pili are capable of retraction, which will bring donor and recipient cells together 19 resulting in close proximity. Indeed, tight conjugative junctions have been observed which have led to the suggestion that cell-to-cell contacts are required for conjugation to take place 20, 21 . However, transfer has also been observed when cells are some distance apart (see detailed discussion below) 22 .

In this review, I will first describe the up-to-date knowledge on each of these complexes and then will discuss the various and sometimes contradictory mechanistic insights that the most recent research shed on the mechanisms of conjugation and type IV secretion.

Building the BOMB molecular biology platform

The essential components of a magnetic bead platform are the beads themselves and a magnet strong enough to immobilise them. Although many life science researchers will be familiar with proprietary beads (e.g., DynaBeads for antibody capture, AMPure beads for size selection), few are aware that they can assemble both beads and magnet components themselves from cheap materials. However, in order to do so, some potentially unfamiliar concepts need to be explained.

Firstly, magnetic beads commonly used for molecular biology come in two major forms—either relatively small (50 nm to 2 μm) particles constructed from a solid ferrite core or larger ferrite-polymer combinations (1–5 μm) [5]. Both bead types work well for nucleic acid purification and manipulation however, their different physical and chemical properties do change their behaviour. For example, the polymer within the larger ferrite-polymer beads effectively lowers the density of the bead so they are less likely to settle out of the suspension during handling steps. The smaller solid-core ferrite beads have a larger relative surface area for binding and can also be easily made in a standard molecular biology laboratory (see BOMB protocols 1–3).

A key aspect of magnetic beads used for molecular biology is that, irrespective of their size, they need to be chemically coated. The first reason for doing this is to provide stability for the bead. Without coating, oxidation of the ferrite would lead to contamination of potentially sensitive samples with iron ions, and the beads would lose their magnetic properties over time. In addition, chemical coating grants additional function to magnetic beads. For example, silica- or carboxyl-polymer coatings are most commonly used because, in addition to providing bead stability, they are relatively chemically inert (silica) or negatively charged (carboxyl), thus facilitating desorption of the negatively charged nucleic acids from the beads during elution steps. Here, we outline a simple protocol for preparation of silica- or carboxyl-coated beads in a standard life science laboratory and production of magnetic racks suitable for their immobilisation.

If we imagine that we're walking on a broad walkway, and that we can't peek over the edge, either at the lateral surface or to the other side, then I don't think there's a way to tell. Suppose the walkway has a handrail, on "both" sides, and you start marking the handrail as you keep your right hand on it. After completing a full loop, you'll be underneath the point across the walkway from where you started. From there, you won't see any mark yet made, because you're underneath the path on which you started. If you keep going, you'll eventually, after another loop, return to the point where you started, and the only mark you see will be on your handrail, not on the handrail across the road to your left.

What has happened here is clearer if you consider what happens when we cut a mobius strip along its midline. Making that cut adds a second edge, producing a normal loop, one edge of which is the original edge of the mobius strip, and the other edge of which is produced by the cut. The walk described in the above paragraph is a walk along one edge of the resulting loop.

If we can reach down and mark the lateral edge of our path, then there's a way to tell. We make regular marks (or a continuous mark) along the lateral edge to our right, and occasionally we check across the path on our left, to see if any marks are on the lateral edge of the path there. Halfway along the complete (2-loop) walk, you would notice marks on the left side, made from "underneath" the path. That would be evidence that you're on a mobius path.

Unraveling the Tangled Complexity of DNA: Combining Mathematical Modeling and Experimental Biology to Understand Replication, Recombination and Repair

How does DNA, the molecule containing genetic information, change its three-dimensional shape during the complex cellular processes of replication, recombination and repair? This is one of the core questions in molecular biology which cannot be answered without help from mathematical modeling. Basic concepts of topology and geometry can be introduced in undergraduate teaching to help students understand counterintuitive complex structural transformations that occur in every living cell. Topoisomerases, a fascinating class of enzymes involved in replication, recombination and repair, catalyze a change in DNA topology through a series of highly coordinated mechanistic steps. Undergraduate biology and mathematics students can visualize and explore the principles of topoisomerase action by using easily available materials such as Velcro, ribbons, telephone cords, zippers and tubing. These simple toys can be used as powerful teaching tools to engage students in hands-on exploration with the goal of learning about both the mathematics and the biology of DNA structure.

5. Proteins

Proteins are linear biopolymers composed of different amino-acid residues covalently linked together by peptide bonds. They play a crucial role in almost all biological processes including cell signalling, catalysing metabolic reactions and structural support. In order to perform their function, most proteins have to fold to a compact 3D structure (native state), which is ultimately dictated by its unique amino-acid sequence.

Many thousands of proteins with a diverse array of structures and functions are known. Due to their structural variation and complexity, proteins have been shown to possess a wide range of intricate topological features (figure 8). Inter-molecular non-covalent interactions can lead to interlocked, oligomeric rings of protein subunits, where the two rings form a Hopf link and therefore become inseparable (figure 8(a)) [133]. In other cases, covalent bonding such as disulphide bonds or metal-side chain interactions can also result in covalent links or knots formed either during or after folding. Figure 8(b) illustrates a Hopf link structure formed as a result of intra-molecular disulphide bonds within each subunit of a dimeric protein [134]. In addition, the recently discovered pierced lasso bundle (PLB) topology is an example of a knot-like motif where the disulphide bond creates a covalent loop through which part of the polypeptide chain is threaded (figure 8(c)) [135]. 'Cysteine knots' can form when a disulphide bond between two segments of a polypeptide chain pass through a ring formed by two other disulphide bonds and their connecting backbone segments (figure 8(d)). Examples include the cyclotide family of naturally occurring plant-based miniproteins and the superfamily of growth factors and toxins [136–138]. In all of these cases, the link or knot is created by a covalent bond or oligomeric structure.

Figure 8. Different types of topologically complex protein structures. In each panel, the protein structure produced using Pymol ( is shown on the left, with a simplified representation of the topology of the system on the right. (a) The crystal structure of bovine mitochondrial peroxiredoxin III forms a Hopf link, PDB code: 1ZYE. In the simplified representation, the blue and red filled circles represent a single chain subunit which associate together to form a higher-order oligomeric ring structure. (b) P. aerophilum dimeric citrate synthase is topologically linked by two intramolecular disulphide bonds (black bars), PDB code: 2IBP. Each protein chain is coloured separately, in this case, blue or teal. (c) A pierced lasso bundle topology of the native structure of leptin, where a disulphide bridge (black bars) creates a covalent loop through which part of the polypeptide chain is threaded, PDB code: 1AX8. (d) The crystal structure of nerve growth factor contains a cysteine knot motif defined by three disulphide bonds (black bars), PDB code: 1BET. (e) The polypeptide backbone chain of E. coli methyltransferase YbeA contains a trefoil knot (31), PDB code: 1NS5. (f) The crystal structure of human phosphatase has a slipknotted topology, PDB code: 1EW2. For (c)–(f), both structures and reduced representations are coloured from blue (N-terminus) to red (C-terminus). Cysteine residues in (b)–(d) are represented as sticks and lines in the structure and simplified representation, respectively.

Complex topologies such as linking or knotting can also be manifested within the protein backbone chain itself. Figure 8(e) illustrates an example of a class of proteins that possess a knotted topological feature in their structures formed by the path of the polypeptide backbone alone [13, 15, 139]. In another case, protein slipknot structures also arise when a protein chain forms a knot but then folds back upon itself to completely untie the knot, thus rendering the structure unknotted when considered in its entirety (figure 8(f)) [140–142]. This section of the review focuses on the structure, function and, in particular, the folding of these types of knotted and slipknotted proteins. Proteins that have knots formed by covalent bonds such as disulphides are not discussed here and readers who are interested in these structures are directed to other publications on these systems [136, 137, 143–146].

5.1. Knotted and slipknotted proteins

For a long time, it was thought that it was highly unlikely, if not impossible, for a polypeptide chain to 'knot' itself to form a functional folded protein. This was, in part, due to the fact that, at that time, no examples of deeply knotted proteins were identified within the protein data bank (PDB) [147]. In this study, a very shallow knot was discovered in carbonic anhydrase by Mansfield [147]. One of the challenges in the search for protein knots was the difficulty in determining whether a knot is present within a complex structure. Thus, for many years, knots in protein structures went undetected. As various computational and mathematical tools were developed to detect and identify knots, it became clear that topologically knotted protein structures do exist, even some with extremely deep knots [24, 26, 148, 149]. Now there are a few web-servers that have simplified the task of knot identification in proteins and can determine quickly whether a structure contains a knot and, if so, what type [150, 151]. In addition, the recent KnotProt database ( created by Sulkowska and co-workers classifies knotted proteins and represents their knotting complexity (knot type and depth of knot) as a 'knotting fingerprint' in the form of a matrix diagram [142, 152, 153]. Matrix diagrams, which are an excellent method for visualising knots and slipknots in proteins, were originally used in the analysis of slipknots in proteins by the Yeates group [140].

To date, over 750 knotted proteins have been discovered within the PDB, equivalent to approximately 1% of all entries [152]. A current list of examples of these structures is provided in table 2. It is worth noting that the KnotProt database is updated regularly [152]. Over the years, a growing number of knotted proteins have been observed in all three domains of life [15, 142, 154]. These include structures that contain a trefoil (31), figure-of-eight (41), Gordian (52) and stevedore (61) knot with three, four, five and six projected crossings of the polypeptide backbone, respectively (figure 9).

Table 2. Examples of knotted and slipknotted proteins. For each fold, the PDB code for the structure of the protein or a typical protein in the family is given. + and − indicates right and left-handed knots and slipknots, respectively.

Protein family or Protein PDB code Knot type
RNA methyltransferase (α/β knot) 1NS5 31 + knot
Carbonic anhydrase 1LUG 31 + knot
SAM synthetase 1FUG 31 + knot
Transcarbamylase fold 1JS1 31 + knot
Sodium/calcium exchanger membrane protein 3V5S 31 + knot
Zinc-finger fold 2K0A 31 − knot
Ribbon-helix–helix superfamily 2EFV 31 − knot
Artificially knotted protein 3MLG 31 − knot
Class II ketol acid reductoisomerase 1YVE 41 knot
Chromophore binding domain of phytochrome 2O9C 41 knot
Ubiquitin C-terminal hydrolases (UCHs) 2ETL 52 − knot
α-haloacid dehalogenase I 3BJX 61 + knot
Alkaline phosphatase 1ALK 31 + slipknot
Thymidine kinase 1P6X 31 + slipknot
Glutamate symport protein 2NWL 31 + slipknot
Sulfatase 4TN0 31 + slipknot
STIV B116 2J85 31 + slipknot
Apoptosis inducing factor 1GV4 31 − slipknot
Sodium:neurotransmitter symporter family 2A65 31 + & 41 slipknot
Betaine/Carnitine/Choline Transporter (BCCT) family 4AIN 31 + & 41 slipknot

Figure 9. Structures of knotted proteins that contain the four different types of knots (31, 41, 52, 61) in the polypeptide backbone. (a) YbeA, a trefoil-knotted (31) methyltransferase from E. coli, PDB code: 1NS5. (b) E. coli class II ketol-acid reductoisomerase, containing the figure-of-eight (41) knot, PDB code: 1YRL. (c) Human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), containing a knot with five projected crossings (52), PDB code: 2ETL. (d) α-haloacid dehalogenase containing a stevedore (61) knot, PDB code: 4N2X. Top panel: ribbon diagrams of the polypeptide chains produced using Pymol ( Lower panel: simplified view of the protein chain showing the knot, generated using KnotPlot ( Both structures and reduced representations are coloured from blue (N-terminus) to red (C-terminus).

Trefoil knots are the most prevalent and simplest type of knot discovered in proteins. The first protein trefoil knot to be identified was that found in carbonic anhydrase—a family of proteins involved in catalysing the reaction of carbon dioxide to hydrogen carbonate and H + [147]. This trefoil, however, is rather shallow as the C-terminus extends through a wide loop by only a few residues. A few years after Mansfield's 1994 study, a much deeper trefoil knot was detected in E. coli S-adenosylmethionine synthetase, an enzyme that catalyses the reaction between methionine and ATP [155, 156]. By far, the largest and most well-studied family of deeply knotted proteins is the trefoil α/β knot fold—a class of methyltransferases (MTases) which are members of the SpoU family [157, 158]. These knotted proteins share common structural features and it is highly likely that all are MTases that catalyse the transfer of the methyl group of S-adenosyl methionine (AdoMet) to carbon, nitrogen or oxygen atoms in DNA, RNA, proteins and other small molecules [159]. In solution, all form dimers with the knotted region comprising part of the AdoMet binding site and forming a large part of the dimer interface [157, 160–163]. Trefoil knots have also been found in two homologues of N-succinylornithine transcarbamylase the AOTCase from X. campestris catalyses the reaction from N-acetylornithine and carbamyl phosphate to acetylcitrulline [164], and SOTCase from B. fragilis promotes the carbamylation of N-succinylornithine [165]. Besides being found in enzymes, trefoil knots have also been identified in Rds3p, a eukaryotic metal-binding protein essential for pre-mRNA splicing [166] and more recently, in the family of sodium/calcium exchanger membrane proteins [152].

More complex knots have also been identified in proteins that catalyse various enzymatic reactions. A deeply embedded, figure-of-eight protein knot has been found in plant ketol-acid reductoisomerases, which are involved in the biosynthesis of branched-chain amino acids [167, 168]. In addition, a Gordian knot has been identified in the family of mammalian ubiquitin carboxyl-terminal hydrolases (UCHs) the proteins are deubiquitinating enzymes that catalyse the cleavage of the isopeptide bond formed between ubiquitin and lysine side chains of protein and other adducts, and thus are involved in the ubiquitin-proteasome system [169–171]. The most complex protein knot known to date is the 61 stevedore knot discovered in DehI, a α-haloacid dehalogenase that catalyses the removal of halides from organic haloacids [154]. Apart from these enzymes, it has been shown that the figure-of-eight knot also exists in the chromophore-binding domain of a red/far-red photoreceptor phytochrome from bacterium D. radiodurans [172, 173].

Slipknotted structures have also been found in a number of proteins (figure 8(f)) [140]. They cannot be identified using the standard methods for knot detection in proteins as, in these cases, the knot becomes undone when the chain is pulled at both termini. As such, it comes as no surprise that these structures had been overlooked until relatively recently. In 2007, Yeates and co-workers first discovered a number of protein slipknots by using an approach based on the fact that slipknots become real knots at some point when the polypeptide chains are shortened [140]. At present, over 450 protein slipknots have been identified [152] and a list of examples of these structures is listed in table 2. It is worth noting that the KnotProt database is the first, and currently only, database that provides details on slipknotted structures [152].

Alkaline phosphatase is the largest family of proteins that contain deep slipknots [15, 140, 152]. In the case of E. coli alkaline phosphatase, 30 residues have to be deleted from the C-terminus before a knotted conformation results. Similar to that of knotted proteins, many of the protein slipknots discovered to date are also found in other enzymes such as thymidine kinases and sulfatases [15, 140, 152]. Interestingly, slipknots have also been found in transmembrane proteins that span the entire cell membrane to which they are permanently embedded [15, 140, 152]. Examples include the families of sodium:neurotransmitter transporters, betaine/carnitine/choline transporters (BCCT) and proton:glutamate transporters [142].

Further details of knotted and slipknotted protein structures can be found in other recent reviews [12, 13, 15, 174] and the KnotProt server [152]. It should be noted that the KnotProt database also provides extensive key information about the biological functions of proteins with knots and slipknots [152].

5.2. Potential roles and implications of the knot and slipknot

Topologically knotted proteins have been found to be conserved across different families [142], suggesting that the knot itself may be advantageous and important to the function of the protein. It has been speculated that a knotted topology could play a key role in increasing catalytic activity or ligand binding affinity (potentially by decreasing dynamics) or enhancing stability (thermodynamic, kinetic and mechanical) of a protein. As yet, relatively little is known about the functional advantages, if any, of these complex knotted structures over their unknotted counterparts. However, various experimental and computational studies have been undertaken to address this question.

Many reports have shown that the knotted regions of knotted proteins play crucial roles in enzymatic activities and ligand binding. As discussed in section 5.1., it has been observed that the knotted regions of the proteins in the α/β-knotted SpoU MTase family comprise part of the active site to which the ligand binds (two examples of α/β knot MTases are illustrated in figure 10(a)) [159–162]. In the case of the N-succinylornithine transcarbamylase, Virnau and co-workers have demonstrated through a computational study that the presence of the knot in the knotted homologue AOTCase may structurally modify its active site and subsequently, may alter its enzymatic activity (in terms of substrate specificity) compared to its unknotted homologue OTCase (figure 10(b)) [149]. In addition, structural studies of the D. radiodurans phytochrome revealed that the deeply embedded knot in the chromophore-binding domain is in contact with the chromophore [172, 173]. A recent study on the conservation of knotting fingerprints in UCHs also showed that there was a correlation between the locations of active site residues and points characterising its knotted topology (i.e. the knotted core) [142]. Despite these examples, there is still little direct experimental evidence that a knotted structure can influence the activity of a protein.

Figure 10. Examples highlighting the potential roles of knots and slipknots. (a) Dimeric structures of the α/β-knot MTases YibK, PDB code: 1MXI (left) and YbeA, PDB code: 1NS5 (right), coloured to show the knotting loop in cyan and the knotted chain in red. S-adenosyl homocysteine, an MTase co-factor, is shown as a stick model. (b) Structures of the knotted section (residues 171–278) of AOTCase with the reaction product N-acetylcitrulline and interacting side chains represented as sticks, PDB code: 3KZK (left), and corresponding (unknotted) section (residues 189–286) in OTCase with the inhibitor L-norvaline (analogous to its L-ornithine ligand) and interacting side chains shown as sticks, PDB code: 1C9Y (right). The knot containsf a rigid proline-rich loop (residues 178–185, coloured red) through which the chain is threaded. (c) Left panel: engineered knotted and unknotted ('superficially knotted') polymers using two different protein constructs. Right panel: first derivative melting curves obtained for the knotted and unknotted polymers. Adapted from [179], by permission of Oxford University Press. (d) Structures of transmembrane proteins LeuT(Aa), PDB code: 2A65 and Glt(Ph), PDB code: 2NWL, where the slipknot loop is coloured cyan and the slipknotted chain in red. Helices are represented as cylinders to ease visualisation. All structures are produced using Pymol (

The question of whether knots have any effect on the conformational dynamics of proteins has also been raised. In the phytochrome protein, it has been noted that the figure-of-eight knot sits where increased rigidity could be important in driving conformational changes that occur when light energy is absorbed by the chromophore [172, 175]. Recent computational approaches using simple lattice models have shown a narrow and less extended native basin for a 52-knotted structure relative to a similar but unknotted one, suggesting enhanced rigidity [176]. However, experimental studies by Andersson et al, which measured 15 N spin relaxation parameters using NMR experiments for the 52-knotted UCH-L1, reported no significant differences between the relaxation properties of the knotted protein relative to unknotted proteins of a similar size [177]. Thus, it remains to be clearly established, particularly experimentally, whether knotted structures can influence the conformational dynamics of a protein.

Much research effort has been undertaken to address the question of whether a knot can provide additional thermodynamic, kinetic or mechanical stability to a protein structure. Sulkowska et al performed coarse-grained simulations of the thermal and mechanical unfolding of the knotted (AOTCase) and unknotted (OTCase) variants of the transcarbamylase-like proteins as well as a synthetic construct of the knotted parent protein rewired so as to remove the knot [178]. In this case, the knotted structure was found to have longer unfolding times than the other two unknotted proteins, which were attributed to topological and geometrical frustration [178]. In an attempt to investigate the potential thermal stabilities of knotted proteins in an experimental study, Yeates and co-workers engineered a knotted and an unknotted ('superficially knotted') polymer [179]. They showed that the knotted chain had a higher thermal stability than the unknotted one (figure 10(c)), although it is important to note that the unfolding in both cases was not fully reversible and therefore only apparent melting temperatures were reported. However, computational studies using Monte Carlo simulations of a simple lattice model using Gō-like potentials showed that a trefoil knot did not have any effect on the thermodynamic stability of a simple protein structure [180]. Instead, it was found that the knot enhances kinetic stability as the knotted protein unfolds at a distinctively slower rate than its unknotted counterpart [180]. Further studies by the same group demonstrated that a more topologically complex protein knot, the 52 knot, clearly enhanced the protein's kinetic stability in comparison to that of a protein containing a 31 knot [176].

The resistance of knotted proteins to mechanical unfolding has been examined by atomic force microscopy (AFM). The first system to be studied was the shallow trefoil-knotted carbonic anhydrase B. In this particular case, an extremely high resistance to unfolding was observed when the protein was pulled from its termini in contrast to a considerably lower resistance when the molecule was pulled from other positions resulting in the untying of the knot [181, 182]. Although these initial studies suggested a dramatic effect of a knot on mechanical stability, the results have not been observed in AFM studies of other knotted systems [175]. In the case of carbonic anhydrase B, recent simulations have shed light on the possible reasons for its remarkable mechanostability [183]. These studies revealed that after an initial, rather limited unfolding event, the knot is wrapped around an inner β-sheet structure in the core of the protein. Thus, the knot is tightened but effectively locally captured by a structural obstacle in the chain. This is aided by the stabilising effects of a zinc ion, which coordinates to the region that becomes entangled by the knot. The simulations explain why in the AFM experiments, the contour length observed is so much smaller than that expected for a fully stretched polypeptide chain containing a tightened knot. In an interesting extension of their initial work, Ikai and co-workers made a tandem repeat of carbonic anhydrase B. Combining AFM with biochemical measurements of activity and binding, they were able to establish that the C-terminal knotted region was essential for activity [184].

The mechanical stability of the 41-knotted phytochrome protein has also been investigated by Bornschögl and co-workers using AFM [175]. In this case, however, they did not observe any enhanced resistance when the knot was tightened as the extension force for unfolding (73 pN) was within the range found for other unknotted proteins. It appears that whether a knot contributes to mechanical stability or not, may depend upon a number of factors including other aspects of the protein's structure and potentially pulling speed/force etc. Several computational studies have suggested that knotting might increase a knotted protein's mechanical stability, thus making it more resistant to cellular translocation and degradation pathways [149, 178, 185, 186]. Again, whether knotting confers any advantageous stabilising effect to a knotted protein over its unknotted counterpart is still inconclusive and thus remains to be tested with more experimental and computational studies.

The significant number of protein slipknots that have now been identified has also posed the question of whether such topologies have any functional or structural role in the protein. In the case of the homodimeric E. coli alkaline phosphatase, Yeates and co-workers engineered cysteine residues at various positions in the protruding loop of the slipknot such that inter-molecular disulphide bonding between the two subunits resulted in a knotted system [140]. Using thermal denaturation, the results showed that the knotted mutants were more thermally stable than either the wild-type or other control mutants. This suggested that the slipknot in the structure may play a role in the enzyme's thermostability [140]. It is also worth noting that the slipknotted B116-like protein is found in a virus that infects thermophilic Sulfolobus archaebacteria [140]. In another study, knotting fingerprint analyses of transmembrane transporting channels from five different families of proteins showed that the slipknotted topology is conserved. This has led to speculations that the slipknot loop, which straps together several transmembrane α-helices, may stabilise their location inside the membrane during their transporter and symporter action [142] (see figure 10(d) for examples of the structures of two slipknotted transmembrane proteins).

5.3. Experimental and computational insights into how knotted and slipknotted proteins fold

The study of how proteins achieve their unique 3D conformation (native state) has been the focus of many researchers in the field of protein folding. For many decades, extensive folding studies focussed on small, monomeric proteins and thus mechanisms of how they fold are now relatively well established [187–191]. These include the framework, nucleation-condensation and hydrophobic collapse mechanisms, which can be viewed as points on a spectrum of a unified mechanism [187, 188]. Current folding theories have shown that small, monomeric proteins, which fold efficiently and rapidly, can achieve their low-energy native configuration from an ensemble of denatured polypeptide chains in a highly cooperative manner and traverse relatively smooth, funneled energy landscapes [192, 193]. However, it is still unclear how these concepts and mechanisms are applicable to larger proteins with more complex topologies including the classes of knotted and slipknotted proteins. Not only do such proteins have to avoid kinetic traps but they also have to overcome significant topological barriers during folding. This section summarises recent developments made towards understanding the mechanisms involved in the formation of these types of complex structures.

5.3.1. Experimental studies on knotted proteins.

Although the elucidation of how knotted proteins fold using experimental approaches remains challenging, in recent years, some significant progress has been made. Most of the experimental folding studies on knotted proteins have focussed on the trefoil-knotted α/β MTases, YibK from H. influenzae and YbeA from E. coli [194–201]. Both proteins are homodimers, which bind to the co-factors AdoMet and S-adenosyl homocysteine (AdoHcy) and contain a trefoil knot at the C-terminus in which at least 40 residues pass through a similarly sized loop (figure 10(a)) [160, 202]. Extensive biophysical techniques have been employed to probe the knotting and folding mechanisms of purified, recombinantly expressed YibK and YbeA. Both unfold reversibly in vitro upon addition of chemical denaturant with a concomitant loss of secondary and tertiary structure [195, 198]. Kinetic studies demonstrated that YibK and YbeA fold similarly via sequential mechanisms that involved one or more monomeric intermediate states and a slow rate-limiting dimerization step [196, 198].

To probe chain knotting events during the folding of YibK and YbeA, Mallam and co-workers constructed a set of knotted fusion proteins in which A. fulgidus This, a stable 91-residue protein, was fused to the N-, C- or both termini of both MTases [201]. This was used as a 'molecular plug' in an attempt to disrupt threading events or to prevent the chain from knotting altogether. Remarkably, these experiments established that both proteins can withstand the fusion of additional domains to both their N- and C-termini and are able to fold to native or native-like states capable of binding cofactor. The fusion proteins created in this study represent some of the most deeply knotted proteins known, the C-terminal fusions requiring some 140 or more residues to pass through a loop to form the knotted native state. Surprisingly, all the fusion proteins showed unfolding and refolding kinetics very similar to the parent MTase giving the first hint that the polypeptide chain might remain knotted even in a highly unstructured chemically denatured state. This was subsequently shown to be the case through in vitro folding experiments on circularized variants of YibK and YbeA, Mallam and co-workers discovered that the denatured ensembles, even in high concentrations of chemical denaturant under which conditions there was little or no secondary or tertiary structure, contained kinetically trapped knotted polypeptide chains [194]. It was then concluded that all the previous in vitro folding experiments on these recombinantly expressed and chemically denatured proteins actually probed refolding from an unfolded but knotted denatured state to a knotted and folded native structure. This unexpected result suggests that there are interactions in the denatured state that kinetically stabilize the knot. Although far-UV CD measurements indicate that there is no significant secondary structure present in the denatured state, recent backbone NMR assignments and chemical shifts of urea-denatured YbeA, show that, in fact, some residual secondary structure still remains under these conditions [203]. The fact that the knot can persist in the denatured state over a long period of time was also confirmed by another group who shared that equilibrium unfolding and refolding transitions of a structurally homologous MTase displayed apparent hysteresis [204]. This behaviour was speculated to be consistent with the uncoupling of the unfolding and untying events of the knotted protein [204]. Recently, single-molecule fluorescence resonance energy transfer (FRET) experiments were performed to characterise the denatured state of TrmD, another trefoil-knotted MTase [205]. Results suggested that the knot was not only retained under denaturing conditions (similar to that of YibK and YbeA) but also slid towards the C-terminus of the polypeptide chain during the unfolding process [205].

Up until recently, there have been no experimental studies into how the knot is first formed from an unknotted linear polypeptide chain. However, with the use of a coupled in vitro transcription-translation system and kinetic pulse-proteolysis experiments, Mallam and Jackson were able to specifically probe folding of nascent chains of YibK and YbeA after they were first synthesised by the ribosome (figure 11(a)) [199]. The results showed that the nascent chains could fold correctly to their trefoil-knotted structure, albeit very slowly. Moreover, a significant lag period between chain synthesis and emergence of a proteolytically stable native state was observed. The results were consistent with the protein knotting and folding from an initially unknotted nascent chain, thus demonstrating that a process associated with the knotting step is rate limiting. Additionally, the GroEL-GroES chaperonin was found to have a dramatic effect on the folding rate of the newly translated polypeptide chains, thus establishing that chaperonins are likely to be important in the post-translational folding of these bacterial knotted proteins in vivo.

Figure 11. Experimental characterisation of the folding of the trefoil-knotted methyltransferases, YibK and YbeA. (a) A schematic representation of the folding and knotting pathways that have been experimentally observed. (b) A schematic diagram illustrating a possible active mechanism for the bacterial GroEL-GroES chaperonin action on the folding of bacterial trefoil-knotted methyltransferase. D, denatured I, intermediate N, native.

Very recently, we have investigated the knotting and folding behaviour of the nascent chains of the different N- and C-terminal This fusions of YibK and YbeA with the use of the coupled in vitro transcription-translation system and kinetic pulse-proteolysis experiments [206]. The results demonstrated that these multi-domain proteins with extremely deep knots can be synthesized in vitro and spontaneously knot without the help of any molecular chaperones, albeit very slowly. In addition, it was concluded that the C-terminus of these proteins is critical to the threading of the polypeptide chain to form the knot, thus providing the first experimental insight as to the mechanism of knotting for this class of bacterial knotted MTase. Further experiments with the GroEL-GroES chaperonin demonstrated that it actively assists the folding of knotted proteins by a mechanism that may involve the unfolding of kinetically trapped unknotted and misfolded intermediates (figure 11(b)). These key observations provide not only vital information into the complex folding pathway of trefoil-knotted proteins but also further insights into how topologically knotted proteins have withstood evolutionary pressures and achieve efficient folding in vivo.

In 2010, the Yeates group engineered an artificially trefoil-knotted protein by covalently linking together two monomers intertwined in the dimeric structure of HP0242 from H. pylori [207]. An in vitro experimental characterisation of this designed knotted protein and an unknotted monomeric variant of the HP0242 dimer was undertaken. Results showed that, although the knotted variant was more stable than the unknotted one, it folded at a considerably slower rate (approximately 20-fold), indicating that knotting, or some event associated with it, is likely rate-limiting.

AFM has also been used to study the mechanical unfolding of the shallow trefoil-knotted carbonic anhydrase B. In this case, the polypeptide chain was found to extend to a distance much shorter than its theoretical stretching length, indicating that the knotted structure is tightened but retained [182, 208]. Similarly, AFM mechanical unfolding experiments on the figure-of-eight knot in the chromophore-binding domain of the phytochrome also resulted in a tightened knot of approximately 17 residues [175]. Although these experiments do not necessarily provide extensive information on the folding pathways of these proteins, they were critical in demonstrating that the knots were present in the structure and in determining the minimum length of polypeptide chain required for knotting.

In addition to the trefoil-knotted proteins described in detail above, the other family of knotted proteins for which there has been any substantial experimental characterisation of their folding pathways are the 52-knotted UCHs [177, 209]. The unfolding of two human UCHs- UCH-L1, a neuronal form of the enzyme, and UCH-L3, ubiquitously expressed in many cell types, have been determined and, in both cases, the in vitro unfolding/refolding with chemical denaturants was shown to be fully reversible [177, 209]. In the case of UCH-L3, equilibrium unfolding data were fitted to a simple two-state model [209] whilst that for UCH-L1 were consistent with a three-state model in which an intermediate state is populated [177]. Using NMR hydrogen-deuterium exchange (HDX) experiments, the intermediate state was characterised indirectly and it was found that the central β-sheet core of the protein remains structured whilst many of the surrounding α-helices have unfolded [177]. Although a more complete analysis of the folding pathway of UCH-L1 has yet to be published, the folding is similar to UCH-L3, such that, both have multiple unfolding and refolding phases that indicate parallel pathways and the population of at least two, metastable intermediate states (Luo et al unpublished results).

5.3.2. Computational studies on knotted proteins.

Many computational studies have shed considerable light on the folding of knotted proteins. Coarse-grained simulations have been excellent at revealing the possible mechanism(s) and generic features of how knotted proteins fold [210, 211]. Wallin et al performed the first such simulation using a Cα model representation of YibK and, similar to experimental studies, observed two parallel folding pathways [210]. They also concluded that specific, non-native interactions involving residues in the C-terminal region of the chain were needed for the protein to knot and fold successfully. In contrast, Sulkowska and co-workers showed that native interactions alone are sufficient for simulating the folding of YibK and YbeA using a coarse-grained structure-based model, although the number of successful trajectories was only 1–2% [211]. These simulations also illustrated that partial unfolding (backtracking) events were needed because the order in which native contacts are formed is critical for the correct folding of the knotted structure and that folding frequently occurred through a slipknotted intermediate (figure 12(a)). Importantly, in the same study, simulations of a rewired, unknotted variant established that there are significant topological barriers in the folding of the knotted structure [211]. Using a similar model, initial results from recent kinetic unfolding simulations of a structurally homologous MTase revealed that unfolding of the protein to a fully unfolded, unknotted state occurs in a stepwise process [204]. In addition, the simulations showed that unknotting of the chain is slow compared to the initial unfolding [199].

Figure 12. Computational simulations of the folding pathways of knotted proteins. (a) Structure-based model used to simulate the folding of trefoil-knotted MTase where the folding route that leads to the native knotted conformation occurs through an intermediate 'slipknot' configuration. Incorrect configurations have to use a 'backtracking' mechanism in order to escape kinetic traps which act as topological barriers. Adapted from [211]. (b) Snapshots taken from the folding simulation of the 61-knotted protein, DehI. Copyright 2010 Bölinger et al [154]. (c) An all-atom structure-based molecular dynamics simulation of the folding pathway of MJ0366. The protein forms a loop with the correct chirality (I), from which it follows two routes to the native state (N): a 'plugging' or 'slipknotting' route. T is an example of how the protein may be kinetically trapped and thus unable to proceed to N. Adapted from [141]. (d) Schematic representations of pulling a trefoil-knotted protein in different points (indicated by the circles) and their resulting final conformations.

Similar computational approaches were also employed in the folding simulations of the 61-knot in DehI [154]. Although the probability of successful folds was low, the study revealed that the complex knotted structure can be formed by a simple tying process. In this case, two unknotted loops, a small loop and a larger loop (which includes a proline-rich unstructured region) are aligned and a knot can be formed by two alternative routes (figure 12(b)) [154]. In the first route, the C-terminus is threaded through the smaller loop (S-loop) via a slipknot conformation before the larger loop (B-loop) flips over the smaller loop. In the other route, the order of the two steps is reversed.

In contrast to very small proteins with simple architectures (which generally have fast unfolding and folding rates), all-atom molecular dynamics (MD) simulations have not been extensively applied to knotted systems, as they are frequently too large for such atomistic approaches to be used. However, it has been possible to use this method in a few cases on small, shallow knotted proteins, such as for MJ0366 from M. jannaschii, one of the smallest trefoil-knotted protein discovered to date [141]. Data from a thermodynamic analysis of the unfolding/folding revealed that the system is three-state, and an intermediate is first formed by twisting of a loop, followed by a rate-limiting step associated with the threading of the C-terminus through the loop. At temperatures near the folding temperature, two folding mechanisms were observed for the formation of the knotted native structure, whereby threading can occur via (i) a plugging route (the C-terminus goes through the knotting loop first) or (ii) the formation of a slipknot (figure 12(c)) [141]. Interestingly, lowering the temperature of the simulation resulted in mechanistic changes. These include a knotting via threading of the N-terminus and the 'backtracking' of misfolded proteins in topological traps. More recently, simulations on VirC2, a protein that has the same fold as MJ0366 but which possesses a deeper knot, also showed that it has a similar free energy profile, suggesting that topology plays a major role in the folding mechanism [212]. A Gō-like potential in which there is minimal energy frustration was also used to simulate the folding of a truncated mutant of another trefoil-knotted MTase [213]. Results from this study suggested a pathway in which the N-terminal region of the protein folds first and that threading of the C-terminus through the structure to form the knot is a late and rate-limiting step [213].

Molecular dynamics simulations were also used to simulate the high temperature unfolding of YibK [214]. The simulations revealed up to four intermediate states on the free energy landscape consistent with the parallel pathways and multiple intermediates observed in experimental studies. In addition, it was found that the denatured state of YibK only untied at very high simulation temperatures, when the C-terminus threads out of the knotting loop via a slipknot conformation. Other unfolding simulations have also been used to investigate the mechanical stability of knotted proteins and the effect of pulling position, pulling speed and temperature on the unfolding/untying of two other MTases [215]. It was shown that pulling the chain at both termini leads to the tightening of the knot whilst pulling at other positions can result in the unknotting of the chain (figure 12(d)).

Various computational studies have also employed Monte Carlo simulations on lattice models using Gō-like potentials to understand the folding mechanism of knotted proteins. In these cases, a potential based on a generic polymer model is used and additional attractive interactions are included for residues that are in contact with each other in the native state. Faisca and co-workers demonstrated that the folding of a model deeply knotted trefoil protein was much slower than a structurally similar but unknotted variant, and that knotting was a late event and concomitant with folding [216]. Using the same model, Soler and Faisca examined the effect of surface tethering on the folding of the system [217]. In this case, it was shown that the mobility of the terminus closest to the knot is critical for successful folding and hindrance results in a decrease in the folding rate and a change in the knotting pathway such that it involves threading of the other terminus. Recently, the same group extended these studies and used the same model to investigate in further detail the effect of knots, knot depth and motif on folding properties of 31-knotted proteins [180]. The results revealed that deeply knotted proteins have a higher probability of retaining their knots in the denatured ensemble, consistent with experimental studies. Furthermore, it was shown that specific native contacts within the trefoil-knotted core are crucial in maintaining the knot in the denatured state, and that threading occurs in the late stages of folding [180]. Most recently, Soler and co-workers extended their studies to investigate the folding mechanism of the more complex 52-knot [176]. Similar to the trefoil knots, it was shown that the chain terminus that is closest to the knotted core is important for the threading movement to form the knot and in no cases was a mechanism that involved the initial formation of a 31-knot observed. However, it was discovered that the probability of concomitant knotting and folding of 52-knotted proteins is significantly smaller than that for trefoil knots as threading to form the 52 knot is a particularly late conformational event [176].

Monte Carlo simulations of a Cα model of trefoil-knotted AOTCase showed that non-native contacts between the C-terminus and other regions in the protein are critical to form the knotting loop through which the chain is threaded [218], consistent with the study by Wallin and co-workers [210]. The importance of non-native interactions in promoting the folding of the native knotted topology of AOTCase and MJ036 was also recently highlighted in simulations employing protein models with different structural resolution (coarse-grained or atomistic) and various force fields (from pure native-centric to realistic atomistic ones) [219]. Again, it appears that these contacts were found to be between the C-terminus and a loop, through which the chain is threaded.

5.3.3. Experimental and computational studies on slipknots.

Numerous simulation studies have shown that a slipknot may be an important intermediate configuration in the folding of knotted proteins [141, 142, 211, 212] and thus, understanding the mechanisms involved in their formation could offer insights into how deeply knotted proteins fold. Using structure-based coarse-grained simulations, Sulkowska and co-workers investigated the folding of thymidine kinase and found that its slipknotted structure can be achieved by a simple 'flipping' mechanism in which a slipknot loop rotates over the unknotted native core of the protein [211]. The rotation of the loop is most likely assisted by the presence of glycine and proline residues in the hinge regions [211]. However, the low success rate of folding events observed suggests that other factors may be needed to overcome the topological barrier or that the barrier is large. The same group extended these studies and used the same model to analyse the mechanical unfolding of the slipknot in the same protein [220]. Weak stretching forces resulted in the smooth untying of the slipknot whilst a metastable intermediate with a tightened knot was observed at sufficiently large pulling forces. It is worth noting that this behavior of slipknotted structures is different to that observed for uniformly elastic polymers [220]. Recently, He and co-workers used AFM to study experimentally the mechanical unfolding of AFV3-109, a protein which has a relatively simple slipknotted structure [221, 222]. Results showed that the slipknot untied and the polypeptide chain was fully extended when mechanical forces were applied at both termini as expected [221]. In contrast, applying forces at the N-terminus and the threaded loop resulted in the tightening of the slipknot into a trefoil knot involving

13 amino acid residues [222]. In both cases, the unfolding process was found to proceed via multiple parallel pathways in either a two- or three-state fashion, and is consistent with a kinetic partitioning mechanism for mechanical unfolding [221, 222].

5.4. Evolution and conservation

Despite the fact that there are now a considerable number of topologically knotted proteins in the PDB, it is worth noting that most proteins are unknotted. This suggests that evolution has, in general, avoided such structures. However, a recent study by Sulkowska and co-workers has established that, when they do occur, that both knotted and slipknotted topologies are conserved across different families despite very low sequence similarity [142]. Unsurprisingly, the parts of proteins which are strongly conserved are found within the knotted core and potential hinge regions which it has been speculated are important in the threading of the chain to form a knot or slipknot [142].

For some families of proteins, where there are a sizeable number of knotted and unknotted variants, it has been possible to undertake a phylogenetic analysis of the sequences, and thereby identify how knotted structures may have evolved from unknotted ancestors. Potestio and co-workers generated a phylogenetic tree of transcarbamylase-like folds [223]. In this case, it was known that some knotted and unknotted variants had different degrees of sequence identity suggesting pathways where structures and therefore sequences had diverged at different times. For example, the two knotted enzymes AOTCase and SOTCase share only 35% sequence identity [224] whilst the knotted AOTCase has 41% sequence identity with unknotted OTCase [225]. Reconstruction of the phylogenetic tree demonstrated that all the knotted homologues populate a sub-branch of the tree and that they differ from unknotted homologues by the presence of additional loop segments [223]. Thus, it has been suggested that some knotted structures have evolved from unknotted ones by the insertion of a 'knot-promoting' loop, which effectively encompasses another part of the chain thus forming the knot.

Loops have also been implicated in the formation of knotted structures from other studies. Virnau and co-workers used computational approaches to show that the knotted transcarbamylase AOTCase possesses a rather rigid proline-rich loop, which is lacking in the unknotted OTCase (figure 10(b)) [149]. Interestingly, the stevedore knot in α-haloacid dehalogenase DehI is also partly formed by a large proline-rich loop that links two unknotted regions within the structure [154].

Using a completely different approach, the group of Yeates have also demonstrated another route to knotted structures through the rational design of a novel knotted structure. In this case, a monomeric knotted protein was created by fusion of C- and N-terminal chains of a homodimer that forms a highly entangled but unknotted structure. This study demonstrated that the genetic fusion and tandem repeat of a gene of an unknotted dimeric protein could lead to trefoil-knotted structures [207].

It is clear that, once formed through some evolutionary pathway, knotted and slipknotted protein structures are highly conserved. However, through both experimental and computational studies, we also know that these types of structures have more complex folding pathways than their unknotted counterparts. This suggests that the knotted and slipknotted motifs within protein families may, in some way, be advantageous and important to either the function, or regulation, of the protein.

5.5. Summary

In summary, both experimental and computational studies have made significant progress in establishing some of the key general features of the folding pathways of topologically complex proteins. In contrast to small monomeric proteins with simple folds, it is clear that proteins with topologically knotted or slipknotted structures have much more complex energy landscapes with many intermediate states and parallel pathways. Computational studies have provided insights into the folding process, which may involve formation of a twisted loop followed by threading via an intermediate slipknot configuration, a plugging route or a 'flipping' mechanism, in which the knotting step may be rate-limiting [141, 211, 226]. In addition, it seems that non-native interactions may play a more important role for these types of structures with complex architectures than for the folding of smaller proteins with relatively simple folds [227–229]. Moreover, the formation of transient misfolded species that results in kinetic traps in the free energy landscape of topologically knotted proteins highly likely requires backtracking events and potentially the action of molecular chaperones so that the native structure can be both rapidly and efficiently achieved [199, 206, 211]. Such a 'frustrated' folding energy landscape is in contrast to the relatively smooth folding funnels proposed for smaller, simpler proteins [192, 230].

A number of recent studies have shown that knotted and slipknotted proteins are conserved suggesting that the knot, or slipknot, potentially play a role in the structure, stability, function or regulation of the protein. Despite this finding, it still has to be unambiguously established whether there are any advantageous properties of a knotted structure over an unknotted one. Indeed, whether there are any chemical or physical properties of such structures that are fundamentally different from unknotted ones. Understanding and identifying such properties will potentially provide key insights for future protein engineering applications and therapeutic developments.

CRISPR had a life before it became a gene-editing tool

WEAPONS OF MASS EVOLUTION Bacteria and archaea armed with CRISPR systems have been at war with viruses for eons. Here, hordes of viruses known as phages assault a bacterium to turn it into a virus-making factory.

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It is the dazzling star of the biotech world: a powerful new tool that can deftly and precisely alter the structure of DNA. It promises cures for diseases, sturdier crops, malaria-resistant mosquitoes and more. Frenzy over the technique — known as CRISPR/Cas9 — is in full swing. Every week, new CRISPR findings are unfurled in scientific journals. In the courts, universities fight over patents. The media report on the breakthroughs as well as the ethics of this game changer almost daily.

But there is a less sequins-and-glitter side to CRISPR that’s just as alluring to anyone thirsty to understand the natural world. The biology behind CRISPR technology comes from a battle that has been raging for eons, out of sight and yet all around us (and on us, and in us).

The CRISPR editing tool has its origins in microbes — bacteria and archaea that live in obscene numbers everywhere from undersea vents to the snot in the human nose. For billions of years, these single-celled organisms have been at odds with the viruses — known as phages — that attack them, invaders so plentiful that a single drop of seawater can hold 10 million. And natural CRISPR systems (there are many) play a big part in this tussle. They act as gatekeepers, essentially cataloging viruses that get into cells. If a virus shows up again, the cell — and its offspring — can recognize and destroy it. Studying this system will teach biologists much about ecology, disease and the overall workings of life on Earth.

But moving from the simple, textbook story into real life is messy. In the few years since the defensive function of CRISPR systems was first appreciated, microbiologists have busied themselves collecting samples, conducting experiments and crunching reams of DNA data to try to understand what the systems do. From that has come much elegant physiology, a mass of complexity, surprises aplenty — and more than a little mystery.

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Spoiled yogurt

The biology is complicated, and its basic nuts and bolts took some figuring out. There are two parts to CRISPR/Cas systems: the CRISPR bit and the Cas bit. The CRISPR bit — or “clustered regularly interspaced short palindromic repeats” — was stumbled on in the late 1980s and 1990s. Scientists then slowly pieced the story together by studying microbes that thrive in animals’ guts and in salt marshes, that cause the plague and that are used to make delicious yogurt and cheese.

None of the scientists knew what they were dealing with at first. They saw stretches of DNA with a characteristic pattern: short lengths of repeated sequence separated by other DNA sequences now known as spacers. Each spacer was unique. Because the roster of spacers could differ from one cell to the next in a given microbe species, an early realization was that these differences could be useful for forensic “typing” — investigators could tell whether food poisoning cases were linked, or if someone had stolen a company’s yogurt starter culture.

Close encounters

Bacteria use CRISPR/Cas as a form of immunity or self-defense against invaders. A bacterium builds a library of genetic material from past invaders so that, if the same invader attacks again, the bacterium and its offspring can disable it.

L. Marraffini/Nature 2015, Adapted by J. Hirshfeld

But curious findings piled up. Some of those spacers, it turned out, matched the DNA of phages. In a flurry of reports in 2005, scientists showed, to name one example, that strains of the lactic acid bacterium Streptococcus thermophilus contained spacers that matched genetic material of phages known to infect Streptococcus. And the more spacers a strain had, the more resistant it was to attack by phages.

This began to look a lot like learned or adaptive immunity, akin to our own antibody system: After exposure to a specific threat, your immune system remembers and you are thereafter resistant to that threat. In a classic experiment published in Science in 2007, researchers at the food company Danisco showed it was so. They could see new spacers added when a phage infected a culture of S. thermophilus. Afterward, the bacterium was immune to the phage. They could artificially engineer a phage spacer into the CRISPR DNA and see resistance emerge when they took the spacer away, immunity was lost.

This was handy intel for an industry that could find whole vats of yogurt-making bacteria wiped out by phage infestations. It was an exciting time scientifically and commercially, says Rodolphe Barrangou of North Carolina State University in Raleigh, who did a lot of the Danisco work. “It was not just discovering a cool system, but also uncovering a powerful phage-resistance technology for the dairy industry,” he says.

The second part of the CRISPR/Cas system is the Cas bit: a set of genes located near the cluster of CRISPR spacers. The DNA sequences of these genes strongly suggested that they carried instructions for proteins that interact with DNA or RNA in some fashion — sticking to it, cutting it, copying it, unraveling it. When researchers inactivated one Cas gene or another, they saw immunity falter. Clearly, the two bits of the system — CRISPR and Cas — were a team.

It took many more experiments to get to today’s basic model of how CRISPR/Cas systems fight phages — and not just phages. Other types of foreign DNA can get into microbes, including circular rings called plasmids that shuttle from cell to cell and DNA pieces called transposable elements, which jump around within genomes. CRISPRs can fend off these intruders, as well as keep a microbe’s genome in tidy order.

The process works like this: A virus injects its genetic material into the cell. Sensing this danger, the cell selects a little strip of that genetic material and adds it to the spacers in the CRISPR cluster. This step, known as immunization or adaptation, creates a list of encounters a cell has had with viruses, plasmids or other foreign bits of DNA over time — neatly lined up in reverse chronological order, newest to oldest.

Older spacers eventually get shed, but a CRISPR cluster can grow to be long — the record holder to date is 587 spacers in Haliangium ochraceum, a salt-loving microbe isolated from a piece of seaweed. “It’s like looking at the last 600 shots you had in your arm,” says Barrangou. “Think about that.”

New spacer in place, the microbe is now immunized. Later comes targeting. If that same phage enters the cell again, it’s recognized. The cell has made RNA copies of the relevant spacer, which bind to the matching spot on the genome of the invading phage. That “guide RNA” leads Cas proteins to target and snip the phage DNA, defanging the intruder.

All stripes of CRISPR

Scientists have divided the array of known CRISPR systems into five types and 16 subtypes based on DNA sequence data. The distribution of types differs in archaea and bacteria.

K.S. Makarova et al/Nat. Rev. Microbio. 2015, Adapted by J. Hirshfeld

Researchers now know there are a confetti-storm of different CRISPR systems, and the list continues to grow. Some are simple — such as the CRISPR/Cas9 system that’s been adapted for gene editing in more complex creatures (SN: 4/15/17, p. 16) — and some are elaborate, with many protein workhorses deployed to get the job done.

Those who are sleuthing the evolution of CRISPR systems are deciphering a complex story. The part of the CRISPR toolbox involved in immunity (adding spacers after phages inject their genetic material) seems to have originated from a specific type of transposable element called a casposon. But the part responsible for targeting has multiple origins — in some cases, it’s another type of transposable element. In others, it’s a mystery.

The downsides

Given the power of CRISPR systems to ward off foes, one might think every respectable microbe out there in the soils, vents, lakes, guts and nostrils of this planet would have one. Not so.

Numbers are far from certain, partly because science hasn’t come close to identifying all the world’s microbes, let alone probe them all for CRISPRs. But the scads of microbial genetic data accrued so far throw up interesting trends.

Tallies suggest that CRISPR systems are far more prevalent in known archaea than in known bacteria — such systems exist in roughly 90 percent of archaea and about 35 percent of bacteria, says Eugene Koonin, a computational evolutionary biologist at the National Institutes of Health in Bethesda, Md. Archaea and bacteria, though both small and single-celled, are on opposite sides of the tree of life.

Perhaps more significantly, Koonin says, almost all the known microbes that live in superhot environments have CRISPRs. His group’s math models suggest that CRISPR systems are most useful when microbes encounter a big enough variety of viruses to make adaptive memory worth having. But if there’s too much variety, and viruses are changing very fast, CRISPRs don’t really help — because you’d never see the same virus again. The superhot ecosystems, he says, seem to have a stable amount of phage diversity that’s not too high or low.

And CRISPR systems have downsides. Just as people can develop autoimmune reactions against their own bodies, bacteria and archaea can accidentally make CRISPR spacers from bits of their own DNA — and risk chewing up their own genetic material. Researchers have seen this happen. “No immunity comes without a cost,” says Rotem Sorek, a microbial genomicist at the Weizmann Institute of Science in Rehovot, Israel.

But mistakes are rare, and Sorek and his colleagues recently figured out why in the microbe they study. The researchers reported in Nature in 2015 that CRISPR spacers are created from linear bits of DNA — and phage DNA is linear when it enters cells. The bacterial chromosome is protected because of its circular form. Should it break and become linear for a spell, such as when it’s being replicated, it contains signals that ward off the Cas proteins.

There are other negatives to CRISPR systems. It’s not always a bonus to keep out phages and other invaders, which can sometimes bring in useful things. Escherichia coli O157:H7, of food poisoning fame, can make humans sick because of toxin genes it harbors that were brought in by a phage, to name just one of myriad examples. Even CRISPR systems themselves are spread around the microbial kingdom via phages, plasmids or transposable elements.

For microbes that lack CRISPR systems, there are many other ways to repel foreign DNA — as much as 10 percent of a microbial genome may be devoted to hawkish warfare, and new defense systems are still being uncovered.


The war between bacteria and phages is two-sided, of course. Just as a microbe wants to keep doors shut to protect its genetic integrity and escape destruction, the phage wants in.

And so the phage fights back against CRISPRs. It genetically morphs into forms that CRISPRs no longer recognize. Or it designs bespoke artillery. Microbiologist Joe Bondy-Denomy, now at the University of California, San Francisco, happened upon such customized weapons as a grad student in the lab of molecular microbiologist Alan Davidson at the University of Toronto. The team knew that the bacterium Pseudomonas aeruginosa, which lives in soil and water and can cause dangerous infections, has a vigorous CRISPR system. Yet some phages didn’t seem fazed by it.

That’s because those phages have small proteins that will bind to and interfere with this or that part of the CRISPR machinery, such as the Cas enzyme that cuts phage DNA. The binding disables the CRISPR system, the researchers reported in 2015 in Nature. Bondy-Denomy and others have since found anti-CRISPR genes in other phages and other kinds of interloping DNA. The genes are so common, Davidson says, that he wonders how many CRISPR systems are truly active.

In an especially bizarre twist, microbiologist Kimberley Seed of the University of California, Berkeley found a phage that carries its own CRISPR system and uses it to fight back against the cholera bacterium it invades, she and colleagues reported in 2013 in Nature. It chops up a segment of bacterial DNA that normally inhibits phage infection.

Of course, in this never-ending scuffle one would expect the microbes to again fight back against the phages. “It’s something I often get asked: ‘Great, the anti-CRISPRs are there, so where are the anti-anti-CRISPRs?’ ” Bondy-Denomy says. Nobody has found such things yet.

Evolution drivers

It’s one thing to study CRISPR systems in well-controlled lab settings, or in just one type of microbe. It’s another to understand what all the various CRISPRs do to shape the ecosystem of a bubbling hot spring, human gut, diseased lung or cholera-tainted river. Estimates of CRISPR abundance could drop as more sampling is done, especially of dark horse microbes that researchers know little about.

In a 2016 report in Nature Communications, for example, geomicrobiologist Jill Banfield of UC Berkeley and colleagues detected 1,724 microbes in Colorado groundwater that had been treated to boost the abundance of types that are difficult to isolate. CRISPR systems were much rarer in this sample than in databases of better-known microbes.

Tallying CRISPRs is just the start, of course. Microbial communities — including those inside our own guts, where there are plenty of CRISPR systems and phages — are dynamic, not frozen. How do CRISPRs shape the evolution of phages and microbes in the wild? Banfield’s and Barrangou’s labs teamed up to watch as S. thermophilus and phages incubated together in a milk medium for hundreds of days. The team saw bacterial numbers fall as phages invaded then bacteria acquired spacers against the phage and rallied — and phage numbers fell downward in turn. Then new phage populations sprang up, immune to S. thermophilus defenses because of genetic changes. In this way, the researchers reported in 2016 in mBio, CRISPRs are “one of the fundamental drivers of phage evolution.”

CRISPR systems can be picked up, dropped, then picked up again by bacteria and archaea over time, perhaps as conditions and needs change. The bacterium Vibrio cholerae is an example of this dynamism, as Seed and colleagues reported in 2015 in the Journal of Bacteriology. The older, classical strains of this medical blight harbored CRISPRs, but these strains went largely extinct in the wild in the 1960s. Strains that cause cholera today do not have CRISPRs.

Nobody knows why, Seed says. But scientists stress that it is a mischaracterization to paint the relationship between microbes and phages, plasmids and transposable elements as a simplistic war. Phages don’t always wreak havoc they can slip their genomes quietly into the bacterial chromosome and coexist benignly, getting copied along with the host DNA. Phages, plasmids and transposable elements can confer new, useful traits — sometimes even essential ones. Indeed, such movement of DNA across species and strains is at the heart of how bacteria and archaea evolve.

So it’s about finding balance. “If you incorporate too much foreign DNA, you cannot maintain a species,” says Luciano Marraffini, a molecular microbiologist at the Rockefeller University in New York City whose work first showed that DNA-cutting was key to CRISPR systems. But you do need to let some DNA in, and it’s likely that some CRISPR systems permit this: The system he studies in Staphylococcus epidermidis, for example, only goes after phages that are in their cell-killing, or lytic, state, he and colleagues reported in 2014 in Nature.

Story continues after graphic

Two roads to travel

Phages don’t always destroy the microbes they invade. Many have two states: They can co-opt a cell’s protein-, RNA- and DNA-making systems to mass produce more of themselves, in what is called the “lytic” cycle, ultimately killing the cell. Or they can insert their genetic material into the host chromosome, to be passively copied each time the cell divides, in the “lysogenic” cycle. That incorporated genetic material can sometimes be useful to the bacterium.

E. Otwell

Sources: Ron Feiner et al/Nat. Rev. Microbiol. 2015 “The Lytic and Lysogenic Cycles of Bacteriophages.” Boundless Biology, August 2016.

Beyond defense

One thing is very clear about CRISPR systems: They are perplexing in many ways. For a start, the spacers in a microbe should reflect its own, individual story of the phages it has encountered. So you’d think there would be local pedigrees, that a bacterium sampled in France would have a different spacer cluster from a bacterium sampled in Argentina. This is not what researchers always see.

Take the nasty P. aeruginosa. Rachel Whitaker, a microbial population biologist at the University of Illinois at Urbana-Champaign, studies Pseudomonas samples collected from people with cystic fibrosis, whose lungs develop chronic infections. She’s found no sign that two patients living close to each other carry more-similar P. aeruginosa CRISPRs than two patients thousands of miles apart. Yet surely one would expect nearby CRISPRs to be closer matches, because the Pseudomonas would have encountered similar phages. “It’s very weird,” Whitaker says.

Others have seen the same thing in heat-loving bacteria sampled from very distant bubbling hot springs. It’s as if scientists don’t truly understand how bacteria spread around the world — there could be a strong effect of far-flung passage by air or wind, says Konstantin Severinov, who studies CRISPR systems at Rutgers University in New Brunswick, N.J.

Invaders with benefits


Sometimes there are rewards when foreign DNA gets into a cell. The ability to evade antibiotics and resist heavy metals has been traced to genes from phages, plasmids and transposable elements.

  • Staphylococcus aureus (top left), Salmonella typhimurium and other disease-causing bacteria have become resistant to antibiotic drugs with the help of resistance genes carried in on plasmids, transposable elements and phage DNA. Multiple genes are often transferred together.
  • Escherichia coli O157:H7 contains Shiga toxins, a gift from phage DNA, among other imported traits that make the bacterium dangerous.
  • Strains of E. coli, Pseudomonas aeruginosa (top right), Bacillus subtilis and others are resistant to heavy metals, such as mercury, arsenic and chromium. These bacteria are found in polluted waters and in hospitals, where heavy metals are used as disinfectants. Plasmids and transposable elements often transferred the resistance.
  • Rhizobium leguminosarum and other rhizobia can pull nitrogen from the air and make it available to plants because of genes on plasmids that the bacteria harbor.

Another weirdness is the differing vigor of CRISPR systems. Some are very active. Molecular biologist Devaki Bhaya of the Carnegie Institution for Science’s plant biology department at Stanford University sees clear signs that spacers are frequently added and dropped in the cyanobacteria of Yellowstone’s hot springs, for example. But other systems are sluggish, and E. coli, that classic workhorse of genetics research, has a respectable-looking CRISPR system — that is switched off.

It may have been off for a long time. Some 42,000 years ago, a baby woolly mammoth died in what is now northwestern Siberia. The remains, found in 2007, were so well-preserved that the intestines were intact and E. coli DNA could be extracted.

In research published in Molecular Ecology in January, Severinov’s team found surprising similarities between the spacers in the mammoth-derived E. coli CRISPR cluster and those in modern-day E. coli. “There was no turnover in all that time,” Severinov marvels. If the CRISPR system isn’t active, why does E. coli bother to keep it?

That quandary leads neatly to what some researchers refer to as an intellectually “scandalous situation.”

In some cases, the genetic sequence of spacers nicely matches phage DNA. But overall, only a fraction (around 1 to 2 percent) of the spacers scientists know about have been matched to a virus or a plasmid. In E. coli, the spacers don’t match common, classic phages known to infect the bacterium. “Is it the case that there is a huge, unknown amount of viral dark matter in the world?” says Koonin — or are phages evolving superfast? “Or is it something completely different?”

Faced with this conundrum, some researchers strongly suspect — and have evidence — that CRISPR systems may do more than defend they may have other jobs. Communication, perhaps. Or turning genes on and off.

But some microbes’ CRISPR sequences do make sense, especially if looking at the spacers most recently added, and others may be clues to phages still undiscovered. So even as they scratch their heads about many things CRISPR, scientists are also excited by the stories CRISPR clusters can tell about the viruses and other bits of DNA that bacteria and archaea encounter and that they choose, for whatever reason, to note for the record. What do microbes pay attention to? What do they ignore?

CRISPRs offer a bright new window on such questions and, indeed, already are unearthing novel phages and facts about who infects whom in the microscopic world.

“We can catalog everything that’s out there. But we don’t really know what matters,” says Bondy-Denomy. “CRISPRs can help us understand.”

Rosie Mestel is a freelance writer based in Los Angeles.

This article appears in the April 15, 2017, issue of Science News with the headline, “The Original CRISPR: Before becoming a famous tool, the gene editor was a weapon in an unending microscopic war.”

Questions or comments on this article? E-mail us at [email protected]

A version of this article appears in the April 15, 2017 issue of Science News.


R. Barrangou et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science. Vol. 315, March 23, 2007, p. 1709. doi: 10.1126/science.1138140.

E.S. Lander. The Heroes of CRISPR. Cell. Vol. 164, January 14, 2016, p. 18. doi: 10.1016/j.cell.2015.12.041.

L.A. Marraffini. CRISPR-Cas immunity in prokaryotes. Nature. Vol. 526, October 1, 2015, p. 55. doi: 10.1038/nature15386.

D. Paez-Espino et al. Uncovering Earth’s Virome. Nature. Vol. 536, August 25, 2016. p. 425. doi:10.1038/nature19094.

4 Applications

4.1 DNA-Based Plasmonic Nanostructures

Chiral molecules typically exhibit different absorption of left- and right-handed circularly polarized light—a property known as circular dichoism (CD), which has been utilized extensively to characterize chiral molecular structures. [ 133 ] When the difference between absorptions of left- and right-handed polarized light is plotted as a function of wavelength, a pattern of peaks and valleys emerges due to the chirality of the underlying molecules that acts as a sort of fingerprint for the molecule. Most media that exhibit a CD signal have a very weak optical response. [ 134 ] However, a weak optical response can be amplified using plasmonic nanostructures. [ 135, 136 ] For example, Kneer et al. showed an enhanced CD response of a DNA origami sheet when it was placed between two gold nanoparticles. [ 137 ]

A plasmon is a quantum of collective free electron gas oscillation in metal, [ 138 ] which can be excited by light of a specific geometry-dependent (resonant) frequency. If the wavelength of the excitation is similar to or longer than the size of the metal particle (the particle size is typically tens to hundreds of nanometers), the plasmon is localized to the particle's surface. When a plasmon is excited, the electron gas in the particle behaves like a simple dipole oscillating parallel to the direction of the oscillating electric field, enhancing the electric field within one wavelength of the particle's surface (the near-field zone) and increasing the particle's cross sections for both scattering and absorption. If another particle with similar resonance frequency is present in the near-field zone, a collective hybrid mode can be formed. Chiral plasmonic modes can form in either handed nanoparticle groups or in chiral-assembled groups of individual resonant nanoparticles. [ 139 ] This kind of optical response is much stronger than those of chiral molecules and is detectable in the visible light regime, which is highly desirable for sensing applications.

Fabrication of chiral plasmonic devices using traditional lithography methods can be expensive and error prone, in particular if the devices have complex 3D geometries. Being robust, precise, and programmable, self-assembly of DNA has allowed the fabrication of complex plasmonic nanostructures in solution. [ 108, 129, 132 ] DNA molecules can either link metal nanoparticles into a prescribed geometrical shape [ 108, 128 ] or serve as a template for metal nanoparticle assembly. [ 129, 131, 140 ] Self-assembled DNA nanostructures have also been used to build entirely metallic nanostructures with designed plasmonic properties. [ 141 ] Note that, in the plasmonic structures discussed here, the DNA serves as a template upon which plasmonic particles are arranged in a chiral manner.

One of the earliest examples of a plasmonic DNA nanostructure was a DNA pyramid that contained four different-sized gold nanoparticles placed at the pyramid's vertices by means of covalently linked DNA strands (Figure 5a). [ 108 ] Govorov and co-workers theoretically showed that gold nanoparticles arranged on such pyramid-shaped objects, as well as on helices, should exhibit a strong CD response in the visible light regime, exceeding the response of natural chiral molecules. Yan et al. experimentally measured the CD response from a pyramid DNA nanostructure, [ 128 ] showing that intensity of the CD spectra can be precisely controlled through spatial arrangement of different types of nanoparticles at the pyramid's vertices (Figure 5b). A pyramid-like arrangement of four nanoparticles was also realized using a sheet-like DNA origami bundle by attaching three gold nanoparticles to one side of the DNA origami sheet and the fourth to the other side, creating a chiral arrangement. [ 140 ] Helical metal–DNA metamolecules were assembled by Kuzyk and co-workers by decorating a 24-helix DNA origami rod with 10 gold nanoparticles arranged in a helical pattern. [ 142 ] The handedness of the nanoparticle arrangement in such structures was found to flip the sign of the CD angle shift in the visible light regime.

Incorporating optically active elements into conformationally dynamic DNA nanostructures makes it possible to modulate the optical activity of such nanostructures by external stimuli. A conformational transition in a DNA nanostructure can be triggered by either changing the nanostructure's environment or by using dynamic DNA nanotechnology design motifs, including lock-and-key DNA strands and toehold-mediated strand displacement. [ 131 ] Schreiber et al. [ 129 ] used helical metal–DNA nanostructures to construct systems that modulated optical response upon drying or rehydration (Figure 5c). In those systems, drying or rehydration altered the conformation of the nanostructures with respect to the surface they were attached to, switching the direction of the CD response from normal to the surface under wet conditions to parallel to the surface upon drying. In a seminal study, Kuzyk et al. assembled two 14-helix DNA origami bundles, each decorated with a gold nanorod, in the shape of a cross and used auxiliary DNA strands to switch the orientation of the bundles from parallel to normal and vice versa. [ 143 ] A similar principle was employed to realize pH-switchable nanostructures, [ 130, 144 ] where pH-modulated affinity of the TAT/CGC triplets in the DNA locking strands (Figure 5d). Coating gold rods with a layer of silver was shown to greatly increase the optical response of the nanorod systems. [ 145 ] Lan et al. [ 131 ] demonstrated switching between more than two chiral states using a structure in which multiple gold nanorods were attached to a V-shaped DNA origami template (Figure 5d). A folded structure could be converted to an extended structure by adding lock-and-key DNA strands (switch 1 in Figure 5e), and also from left-handed to right-handed conformations via a toehold-mediated strand displacement reaction (switch 2 in Figure 5e). In another approach, [ 146 ] the mutual orientation of several metal nanorods attached to multiple layers of DNA origami rods and sheets was controlled by means of blocker and activator strands, realizing eight distinct plasmonic stereoisomers.

Conjugating metal nanoparticles to a DNA scaffold has made it possible to amplify changes in the CD response caused by molecular interactions for biosensing applications. One such sensor was described by Ma et al., [ 132 ] where self-assembly of gold nanorods was mediated by hybridization of the analyte strand to strands conjugated with the nanorods (Figure 5f). In the assembly, connecting one nanorod to the other via DNA duplexes was found to rotate one nanorod with respect to the other, giving the system a CD response. The amplitude of the CD response was found to increase with the concentration of the DNA analyte and allow detection of the DNA analyte present at attomolar concentrations.

4.2 DNA Spintronics

The intrinsic curvature of a chiral molecule ensures that, as an electron moves through it, an effective magnetic field is generated. This magnetic field acts on the magnetic moment of the electron, and as a result, the tunneling probability through the chiral molecule differs for electrons with opposite spin. This phenomenon is referred to as chiral-induced spin selectivity (CISS), [ 148, 151, 152 ] schematically shown in Figure 6a. The physical origins of CISS can be traced back to the spin-orbit coupling, which is a relativistic effect that arises from the magnetic torque exerted on the electron when it is orbiting around the nucleus. [ 153, 154 ] When considering a 2D material with a broken symmetry, such as an interface, the currents of electron and spins become coupled through the Rashba effect, [ 153, 154 ] which allows for manipulation of spin degrees of freedom via electric field.

Experiments have shown that dsDNA molecules can produce a measurable CISS effect [ 148, 149 ] (Figure 6b). In these experiments, thiolated ssDNA molecules were absorbed onto a Ni substrate while complementary ssDNA strands were bound to gold nanoparticles. Upon hybridization of the stands, Ni–dsDNA–Au junctions were formed and their electrical conductivity was probed using AFM with a conductive tip operating in a contact mode. [ 148, 149 ] A permanent magnet was used to magnetize the Ni substrate, creating two conduction bands in the Ni substrate: one band corresponding to the electrons with spin aligned with the magnetic field and the other aligned opposite to the magnetic field. The resulting IV curves are shown in Figure 6b, bottom. When the spin orientation of the Ni substrate aligns with the favored transmission direction through the chiral molecule, the current is higher compared to when the Ni substrate is magnetized in the other direction. Note that the I–V curves are symmetric, which means the opposite-spin electrons flow from gold to Ni when an opposite bias is applied. We note further that, in DNA spintronics, tunneling occurs between the aromatic orbitals of nucleobases however, chiral molecules without aromatic groups also exhibit this effect. This kind of spin selectivity can happen anywhere a chiral potential exists.

A potential application of DNA's spin selectivity effect is electrochemical water-splitting (Figure 6c). The separation of water, which is spin singlet (containing no unpaired electrons), into H2 (singlets) and O2 (naturally in a spin triplet state, with a total spin of one) is spin forbidden, and thus, this process is slow. [ 147 ] Spin selection can help accelerate this process. In the experiments performed by Mtangi et al., [ 150 ] CdSe nanoparticles (red) were connected to TiO2 nanoparticles, and then the TiO2 nanoparticles were attached to the fluorine-doped tin oxide (FTO) conducting electrode, serving as the anode. The Pt electrode served as the cathode, and hydrogen was produced from that side. Because of the presence of chiral molecules, electrons of only one spin state could be transferred from CdSe to titania. Thus, the spin orientation of holes in the CdSe nanoparticles was well defined. Since the oxygen atoms had the same unpaired spin, the probability of forming O2 was much higher than in a system containing no chiral molecules. [ 150 ]

Because of their switchability and long coherence time, spin systems are promising candidates for storing quantum information. Utilizing CISS to build a spin memory device is also possible, and has been experimentally realized for several (but not nucleic acid) chiral molecules. [ 155-157 ] Figure 6d illustrates the design of a CISS-based memory, which could potentially use dsDNA as a CISS filter molecule. The left panel of Figure 6d depicts an electrical memory, written and read out by the electric current, as conceived by Koplovitz et al. [ 157 ] The ferromagnetic nanoparticle (red) is first magnetized in the direction anti-aligned with the spin direction favoring transfer from substrate to nanoparticle. To write in the memory, a high cross-molecule voltage is applied. Only one spin is allowed to pass through the chiral molecule to the substrate, and, thus, the nanoparticle's spin is flipped. To read out the information, a lower voltage is applied. Because the written bits' spin states are already occupied, the unwritten bits will have a lower resistance than the written bits. The right panel of Figure 6d shows a design of a single bit of optical memory, driven by circularly polarized light. When the nanoparticle is excited by light, the charge oscillates with the electric field, and creates a net spin-transfer torque between the excited quantum dot and the ferromagnetic substrate, leaving a local magnetization in the substrate. [ 147, 155 ] Ben et al. were able to read out this type of memory by measuring the Hall voltage across the substrate. [ 155 ] While these two types of spin-selective memories are silicon-compatible, erasing information requires chemical replacement of the molecules with their enantiomers, which can be a highly inefficient process. Since methods for switching a DNA origami's chirality have already been developed, DNA origami constructs could potentially play a role in the future development of this type of memory device.

4.3 Other Emergent Application Areas

The control and customizability of DNA nanostructures makes them an attractive prospect for targeted drug-delivery. Specifically, DNA origami-based assemblies have served as vehicles for the delivery of anticancer drug doxorubicin (Dox), which intercalates between DNA bases, to infected cells. [ 158, 160, 161 ] In one such study [ 158 ] two rod-like DNA-origami nanostructures with different global designs (Figure 7a) were used to deliver Dox to human breast cancer cells. The globally twisted structure was found to release the drug more slowly, compared to the straight origami-based structure or freely diffusing Dox (Figure 7b). Thus, the kinetics of the drug release appears to be controlled by the global twist of the nanostructure, which open interesting possibilities for regulating drug release dynamically or conditioning the release speed on the presence of specific biomarkers.

Information could be stored or displayed with nanoscale precision using DNA origami as a building material. DNA origami's spin-selectivity could be harnessed to build an array of bits, which could potentially serve as a memory device. Previous studies [ 155, 157 ] used an array of short α-helical peptides to fabricate such a device (Figure 6d). Another potential application is an actively-switchable display (Figure 7c), which could be constructed using nanoparticles arranged using DNA origami into a chiral pattern to provide a strong CD response. When right-handed light shines along the helical axis of a left-handed helix, much of the light will be absorbed. [ 129 ] If the same helix is rotated so that its helical axis is orthogonal to the light, the absorption will decrease. It was recently demonstrated that a DNA origami bundle can be reversibly switched between upright and flat configurations using an electrode. [ 162 ] This mechanism could be used to electrically switch absorption states to make a display.

Last but not least, chiral nanostructures can be instrumental for delivery of individual biomolecules, such as DNA strands or unfolded proteins. Figure 7d illustrates one such possibility, where a spiral step defect in a multi-layer graphene membrane guides the delivery of a single DNA strand to the center of the spiral. [ 159 ] Molecular dynamics simulations and AFM experiments have established that the displacement of an adsorbed molecule over a step defect has a directional anisotropy and that such a molecule is much more likely to move along the step-defect edge than across it. Thus, an external force that switches direction in a pattern that matches the chirality of the edge defect structure will bring the molecule to the center of the structure regardless of where the molecule is adsorbed, whereas applying the external force in the opposite chirality pattern will move the adsorbed molecule away from the center of the structure.


In this paper, we have presented synthetic gene circuits for spatial patterning that are simple in design and construction but are robust and tunable in functioning. The circuits required only minimal DNA parts for assembly, but produced desired patterns without the need for architectural fine tuning. In addition, they were robust against the perturbations of various environmental factors (e.g. media composition, pH value, and temperature) and the variations of cellular contexts (e.g. the host organism and plasmid copy number). Moreover, although robust, the circuits were highly tunable to designed regulatory signals. Conferred by its robustness and tunability, the circuits further allowed us to establish predictable spatial structures of synthetic bacterial communities and to create controllable arrays of cellular grids and spots in space. Compared to other examples constructed to control spatial band behaviors ( 12, 17– 19), this study provides a simple and yet reliable solution to program robust and tunable spatial patterns that can be harnessed to create complex structures of populations. Circuit fragility is one of the major hurdles in synthetic biology, hampering the construction of complex networks for advanced functionalities and the applications of synthetic biology in real-world settings ( 10, 31– 34). To address the challenge, developing new design principles and methodologies have been the major efforts. This study demonstrates that deep mining of the untapped capacities of natural systems, such as the dual functionalities of nisin, can be an alternative strategy to build robustness. Importantly, with appropriate mining, implementation of desired functionality may not require complex circuit construction and optimization.

This work advances our capacity in controlling microbial communities and sensing environmental signals. It may also benefit the development of tissue engineering and biomaterial fabrications that require spatiotemporal control of signaling molecules. In addition, as the band-pass behavior of the engineered circuits is commonly occurring in nature, the study provides insights into the fundamental cellular coordination processes of natural systems, such as microbial community assembly and embryo development.

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