Pore translocation of knotted DNA rings
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Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved March 7, 2017 (received for review January 25, 2017)

Significance
Pore translocation, the driven passage of molecules through narrow channels, has become an important tool for probing DNA properties. In a recent breakthrough experiment, this technique was used to detect knots that form spontaneously in DNA filaments and can hence impact their in vivo functionality. Here, by using an accurate model, we simulate the translocation of knotted DNA, expose its unexpectedly rich phenomenology, and clarify the implications for experiments. We show that knot translocation occurs in two possible modes, depending on the knot initial position and size. These properties also account for the typically late occurrence of the knot passage event. Finally, the passage duration is found to depend more on the translocation velocity of the knot than its size.
Abstract
We use an accurate coarse-grained model for DNA and stochastic molecular dynamics simulations to study the pore translocation of 10-kbp–long DNA rings that are knotted. By monitoring various topological and physical observables we find that there is not one, as previously assumed, but rather two qualitatively different modes of knot translocation. For both modes the pore obstruction caused by knot passage has a brief duration and typically occurs at a late translocation stage. Both effects are well in agreement with experiments and can be rationalized with a transparent model based on the concurrent tensioning and sliding of the translocating knotted chains. We also observed that the duration of the pore obstruction event is more controlled by the knot translocation velocity than the knot size. These features should advance the interpretation and design of future experiments aimed at probing the spontaneous knotting of biopolymers.
How filamentous molecules behave when driven through a narrow pore is one of the classic, yet still open questions in polymer physics. The problem has important applications for single-molecule manipulation techniques, including the sequencing of single-stranded DNA filaments (1⇓⇓⇓⇓⇓⇓–8), and is relevant for fundamental research as well, particularly for biological systems where the processing of DNA (9, 10), RNA (11), and protein chains (12) often depends on their active translocation through narrow pores.
Because knots are statistically inevitable in long polymers and biopolymers (13⇓⇓⇓⇓⇓⇓–20), a relevant question is how such forms of entanglement affect pore translocation (21⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓–34).
Very recently, an important advancement in this research field was made by Plesa et al. (34) who succeeded in devising an advanced single-molecule experiment where double-stranded DNA was translocated through a solid-state nanopore in carefully controlled conditions. The DNA filaments were sufficiently long to be spontaneously knotted in a sizeable fraction of the equilibrium population. The pore diameter, 10–20 nm, was purposely chosen to be smaller than the DNA persistence length,
Here, to advance the understanding of the process and its relationship with DNA knotting in equilibrium, we present a detailed study based on molecular dynamics simulations of an accurate mesoscopic DNA model. Specifically, we consider equilibrated knotted DNA rings of 10 kbp represented with the oxDNA model of refs. 36⇓⇓–39 and use Langevin dynamics to simulate their driven passage through a 10-nm–wide pore. Such a theoretical and computational framework allows us to investigate the translocation process and the geometry–topology interplay with unprecedented structural and dynamical detail.
Our main findings are the following. First, we observe that there is another mode of knot translocation besides the one that has been considered so far. Second, the passage of the entangled region through the pore is largely controlled by the positioning of the knot on the ring and its velocity at the time of translocation. As a consequence, pore obstruction events associated to knot passage are brief and mostly occur at late translocation stages. Finally, these properties, which are in good overall accord with single-molecules experiments, are recapitulated with a schematic interpretative model that can also be used for predictive purposes.
Results and Discussion
System Setup.
We carried out various Langevin dynamics simulations of knotted DNA rings translocating through a nanopore embedded in a slab (Fig. 1). The rings, modeled mesoscopically with oxDNA (36⇓⇓–39), were 10 kbp long and were nicked, to allow relaxation of torsional stress, as in typical experiments (34, 40). The translocation is driven by a longitudinal electric field exerting a force of 0.2 pN on each nucleotide inside the pore. For simplicity, we neglect the action of the field outside the pore (41, 42) that, in actual realizations, can facilitate the capture and pore insertion of the knotted chains (43⇓–45). The pore is 10 nm wide and 10 nm long, so that each of the two dsDNA filaments inside it (
Typical configuration of a knotted double-stranded DNA ring translocating through a nanopore. Inset shows the knot approaching the pore entrance. The ring is 10 kbp long and is modeled with oxDNA. The pore, which is 10 nm wide, is embedded in an impenetrable 10-nm–thick slab. A translocating force of 0.2 pN is applied to each nucleotide inside the pore.
The translocation dynamics were studied for 50 different equilibrated knotted DNA configurations. These were generated with a Monte Carlo scheme applied to a coarse-grained DNA model and were subsequently refined and relaxed with the oxDNA model. All configurations featured a trefoil or
Translocation Dynamics Overview.
For a first, general characterization of the process we profiled the translocated fraction of the chain,
(A) The time required to translocate a fraction
Fig. 2B shows, instead, the so-called waiting time (47),
The first part of the curve, for
At
Statistics of Knot Translocation Events.
Inspired by experiments, we detect the passage of the knot through the pore by monitoring the degree of obstruction of the latter. During such an event, in fact, the pore lumen must accommodate up to four double-stranded filaments, instead of the usual two (sketches in Fig. 3A).
(A) Time evolution of the chain fraction that is inside the pore,
We accordingly monitored the time evolution of the chain fraction inside the pore,
Various observables of interest, related to those monitored in the experiments of ref. 34, can be derived from the analysis of the
The key features are two. First, the distribution of
Probability distribution of the normalized time of knot passage,
This consistency of the experimental and theoretical distributions for
Knot Translocation Modes.
As we discuss, both the position of the knot along the chain contour and its size affect the
A particularly intriguing relationship is found between the pore obstruction duration,
The analysis of the trajectories showed that the distinct arms in the diagram of Fig. 4A originate from two different modes of knot translocation, as described below.
(A) Scatter plot of the knot length when the pore obstruction event starts,
In the first mode the knot is tight and localized on one of the two translocating filaments (Fig. 4D). This is the most intuitive type of knot passage and, in fact, it was the mode of choice used in ref. 34 to interpret the experimental data on
Our results vividly confirm the significant occurrence of tight knots. Indeed, we observe that the average knot length at the passage event is about 54 nm, which is in full accord with the estimate of Plesa et al. (34). This knot length is reached independently of the initial one due to tightening of the knot caused by the propagating chain tension (Fig. 4B). We also note that the
The second, and new mode is associated to the green points in Fig. 4. It involves knots that span a significant portion of the ring, consistent with the theoretical results of ref. 58 on DNA chains of comparable size, which indicated that the most probable knot length is about 2,200 bp. In fact, these knots experience significantly less tightening during translocation than those discussed above (Fig. 4B). Intriguingly, these knots are large and yet their pore-obstruction times are not at all dissimilar from the tight knot case discussed before.
This conundrum is solved by considering the actual conformation of such rings when the knot is presented at the pore entrance. A typical configuration is shown in Fig. 4C. The accompanying sketch clarifies that the knotted portion now spans the entire cis part of the ring. This is quantitatively shown in the semilog plot of Fig. 4E, where we observe that for this class of knots, the relative chain fraction occupied by the knot is
However, a significant obstruction of the pore occurs only when the region accommodating the essential crossings passes through it. As seen in Fig. 4, this region is typically small, involving 123 bp (42 nm) on average upon entering the pore (and is slightly reduced when fully inside it).
It is therefore this short, essentially entangled portion of “double-filament” knots, and not their entire contour lengths,
To our knowledge, the possible occurrence of a second mode of translocation, although rather natural a posteriori, has not been considered or foreseen in previous translocation studies, either for dsDNA ring experiments or for simulations of linear, open chains where it can also occur if translocation starts from inside the knot loop region.
Note that, because the essentially entangled region is comparable in size to the tight, single-filament knots, the two modes of translocation cannot be distinguished from the sole analysis of
(A) Model estimate of relative percentage of single- and double-filament knots in DNA rings of different length. The estimate considers the length and positioning of the knotted region (highlighted in red in the sketches) with respect to the point (marked with a cross) antipodal to the root (marking the point where translocation initiates). (B) The same model is used to predict the translocated chain fraction at the time of the pore obstruction event,
We conclude the analysis of the data in Fig. 4A by discussing the second notable feature, namely the lack of a noticeable correlation between the pore obstruction duration,
This point is clarified by the plot of Fig. 4F, which shows the relationship between
This observation might be harnessed to extract further knot-related properties from
Interpretative Model.
We now consider the origin of the two different translocation modes and of the skewed distribution of the knot position at passage time,
Both aspects are best illustrated with the following schematic model of the translocation process. In this purposely simplified scheme we assume that the rooting point where the translocation process initiates is equally likely to lie anywhere on the ring contour. We also assume that tension propagates in the same way along the two ring arms departing from the root, so that they meet at the antipodal midpoint, that is, at the point at half ring contour length from the root.
The main discriminator for the two translocation modes is whether the knot is entirely located on only one of the two arms or whether it straddles the antipodal midpoint and hence spans both arms (sketches in Fig. 5).
In the former case the sliding of the progressively tensioned ring arm causes the knotted region to tighten toward the distal knot end (i.e., the end that is farthest from the rooting point) while it is dragged to the pore (Movie S3). The tightened single-filament knot will then pass through the pore.
Instead, when the knotted region straddles the antipodal midpoint, the knot will be pulled from both sides and will be dragged toward the pore by both tensioned arms. Such double pulling typically causes the essential crossings to become interlocked, trapping the knot in a moderate degree of tightening (Movie S4). The pore obstruction event is then associated to the passage of the essential crossings.
These two cases are directly associated to the two different translocation modes highlighted in Fig. 4, so much so that the two sets in Fig. 4 were not assigned from an a posteriori supervised inspection of the trajectories, but rather a priori based on the aforementioned distinction. In fact, the two sets precisely correspond to knots that span a single ring arm or both ring arms at the beginning of translocation. The neat separation of the two sets of points in Fig. 4 A, B, E, and F supports the viability and usefulness of such a discriminatory criterion.
The same criterion can be also used to estimate how the relative incidence of single- vs. double-filament entanglement varies with ring length. We considered an ensemble of Monte Carlo-generated rings of length 10 kbp, 20 kbp, and 50 kbp; picked a rooting point randomly along their contour; and then located the knot on the ring, which we considered open in correspondence to the rooting point. The rings were next assigned to one of the two classes based on whether the knotted region straddled the antipodal midpoint or not. The results, given in Fig. 5A, show that the incidence of single-filament entanglement increases steadily with chain length and goes from 35% for 10 kbp to 70% for 50 kbp. Based on this result, which reflects the interplay of knot and chain lengths (35, 58⇓⇓–61), we speculate that most of the knot passage events detected in the experiment of Plesa et al. (34) pertained to single-stranded entanglements, as implicitly assumed by the authors.
The same schematic framework can account for the qualitative features of the distribution of
The resulting probability distributions for
The above modeling scheme neglects the possibility that tight knots may slide on the filament contour. As was clarified in the theoretical study of refs. 27, 30, and 31, such sliding can occur for fully flexible chains and, in fact, make it possible for individual knotted filaments to fully translocate through very narrow pores, as long as the driving force is not high enough to cause jamming (30, 62, 63). As a matter of fact, we observed the same knot sliding phenomenology for the present dsDNA system too (Movies S1–S3).
To account for such a sliding effect for single-filament knots, we accordingly adjusted the model. Specifically, we assumed that
Conclusions and Perspectives
It is only very recently that innovative single-molecule techniques have made it possible to detect knots in dsDNA chains driven through nanopores (34). On the one hand, this gave a striking demonstration of spontaneous knot formation in linear and circularized DNA. On the other hand, it also helped unveil a rich and complex phenomenology that, although expectedly relevant for the in vivo processing of DNA filaments, is still largely unexplored.
Here, to advance the understanding and characterization of such phenomenology, we studied theoretically the pore translocation of knotted DNA rings, using an accurate coarse-grained model for DNA and stochastic molecular dynamics simulations.
We find good agreement with the experimental data, particularly regarding the remarkably brief duration of pore-obstruction events associated to the passage of the knot. By profiling the dynamical evolution of the knotted DNA rings we expose unexpectedly rich properties of the process that cannot be directly accessed in current experiments.
First, we found that translocation of the knotted region can occur in two qualitatively different modes, depending on whether the knot is dragged to the pore by only one of the ring arms or both. In the latter case, knots are typically not tight, and yet we find that the pore obstruction time can be small (as in experiments) because the essential crossings of the knot coalesce in a short region.
Second, we found that the sliding and tensioning of the translocating knot causes the same bias toward late knot passage events found in experiments and heretofore unexplained. We finally show that one of the key determinants of the pore obstruction duration is the initial positioning of the knot along the chain and suggest how this effect might be deconvolved in experimental measurements for a more precise determination of the length of the region accommodating a knot or its essential crossings. In particular, the occurring phenomenon of knot sliding might give an important contribution. This might be exploitable in future experiments, along with chain length and pore size variations, to discriminate the two modes. Further relevant avenues include the impact on pore translocation of complex topologies such as composite knots, which have so far been characterized for flexible chains only (30), as well as the geometry–topology interplay in DNA rings that cannot relax supercoiling and torsional stress (64).
This first theoretical account provides a detailed and physically appealing insight into the phenomenology of knotted dsDNA pore translocation. It provides a valuable and transparent interpretative framework for available experimental data while pointing out specific directions for new experiments as well as theoretical ones aimed at better understanding the implication of intrachain entanglement for the in vivo processing of DNA and possibly other biopolymers too.
Materials and Methods
System Setup and Simulation Details.
For an accurate, mesoscopic description of dsDNA we used the oxDNA model (36⇓⇓–39). In this model, nucleotides are treated as rigid and described by three-interaction centers. The potential energy includes terms that account for the chain connectivity, bending rigidity, base pairing, screened electrostatic interactions, and stacking effects. These terms are parametrized to reproduce the salient structural and equilibrium properties of nucleic acids filaments at various values of the system temperature and salt concentration, here set to
The initial conformations of the 10-kbp–long DNA rings were obtained by mapping the oxDNA model on top of knotted, coarse DNA rings sampled with a topologically unrestricted Monte Carlo scheme (46). These initial configurations, which model those obtained experimentally by circularization of linear DNA with so-called sticky ends, were then nicked and primed at the entrance of the 10-nm–wide pore, which was embedded in a 10-nm–thick impenetrable slab. The translocation process, simulated with Langevin dynamics using the oxDNA software package, was driven by applying a force of 0.2 pN to each nucleotide inside the pore. Because the average number of nucleotides occupying the pore at any given time is 120 (60 bp), the average total driving force is 24 pN (12 pN on each of the two double-stranded filaments), which is about equal to that used in experiments. The constant-temperature (
(A) Typical configurations obtained by the Monte Carlo sampling in ref. 46 where 10-kbp DNA rings were discretized at the 10-nm level. These configurations were next intermediately fine-grained as chains of beads with 0.34-nm spacing. (B) These chains, associated with a nominal persistence length of 50 nm, were relaxed for
Observables.
The passage of a knot, or of its essential crossings, through a pore was revealed by monitoring the number of nucleotides inside the pore and detecting increases by more than 30% from the baseline value, which is about equal to 60 bp. This threshold criterion, which was validated by supervised visual inspection, was also used to establish the duration of the time interval associated to the passage of the knot through the pore.
For each instantaneous configuration, the location of the knot was identified with a bottom–up search. Specifically, we used the stochastic search scheme of ref. 65 to identify the shortest portion of the ring that, after suitable closure, has the same topology as the original ring. The search is limited to the trans or cis parts of the rings, respectively, depending on whether the knot has or has not already translocated through the pore.
Acknowledgments
We thank Flavio Romano and Lorenzo Rovigatti for technical advice on the use of the oxDNA package. We acknowledge support from the Italian Ministry of Education, Grant PRIN 2010HXAW77.
Footnotes
- ↵1To whom correspondence should be addressed. Email: michelet{at}sissa.it.
Author contributions: A.S. and C.M. designed research; A.S. performed research; A.S. and C.M. analyzed data; and A.S. and C.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1701321114/-/DCSupplemental.
References
- ↵.
- Deamer D,
- Akeson M,
- Branton D
- ↵
- ↵.
- Agah S,
- Zheng M,
- Pasquali M,
- Kolomeisky AB
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵.
- Liu LF,
- Perkocha L,
- Calendar R,
- Wang JC
- ↵
- ↵
- ↵
- ↵.
- Rybenkov VV,
- Cozzarelli NR,
- Vologodskii AV
- ↵.
- Sułkowska JI,
- Rawdon EJ,
- Millett KC,
- Onuchic JN,
- Stasiak A
- ↵
- ↵.
- Jackson SE,
- Suma A,
- Micheletti C
- ↵.
- Arsuaga J,
- Vázquez M,
- Trigueros S,
- Sumners D,
- Roca J
- ↵.
- Arsuaga J, et al.
- ↵
- ↵.
- Marenduzzo D, et al.
- ↵
- ↵
- ↵
- ↵.
- Marenduzzo D,
- Micheletti C,
- Orlandini E,
- Sumners DW
- ↵.
- Szymczak P
- ↵.
- Suma A,
- Rosa A,
- Micheletti C
- ↵.
- Narsimhan V,
- Renner CB,
- Doyle PS
- ↵.
- Rieger FC,
- Virnau P
- ↵.
- Wojciechowski M,
- Gómez-Sicilia À,
- Carrión-Vázquez M,
- Cieplak M
- ↵.
- Plesa C, et al.
- ↵
- ↵
- ↵
- ↵
- ↵.
- Snodin BE, et al.
- ↵
- ↵.
- Farahpour F,
- Maleknejad A,
- Varnik F,
- Ejtehadi MR
- ↵
- ↵
- ↵
- ↵
- ↵
- ↵.
- Ikonen T,
- Bhattacharya A,
- Ala-Nissila T,
- Sung W
- ↵.
- Plesa C,
- van Loo N,
- Ketterer P,
- Dietz H,
- Dekker C
- ↵.
- Sakaue T
- ↵
- ↵.
- Sarabadani J,
- Ikonen T,
- Ala-Nissila T
- ↵.
- Kantor Y,
- Kardar M
- ↵.
- Chatelain C,
- Kantor Y,
- Kardar M
- ↵
- ↵
- ↵.
- Ikonen T,
- Bhattacharya A,
- Ala-Nissila T,
- Sung W
- ↵
- ↵.
- Dai L,
- Renner CB,
- Doyle PS
- ↵.
- Dommersnes PG,
- Kantor Y,
- Kardar M
- ↵
- ↵.
- Caraglio M,
- Micheletti C,
- Orlandini E
- ↵
- ↵.
- Narsimhan V,
- Renner CB,
- Doyle PS
- ↵.
- Racko D,
- Benedetti F,
- Dorier J,
- Burnier Y,
- Stasiak A
- ↵.
- Tubiana L,
- Orlandini E,
- Micheletti C
- .
- Suma A,
- Orlandini E,
- Micheletti C
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