Patent Application
20250084407
The present invention relates to a method for the construction of single stranded DNA catenanes at the kilobase length scale. The catenanes can be used for the assembling scaffolds of covalently connected DNA origamis.
Topologically linked molecules such as catenanes and rotaxanes are challenging and fascinating targets in supramolecular chemistry [1]. They achieved particular prominence after Sauvage's demonstration of the templated approach to molecular catenane synthesis in 1983 [2, 3] with Stoddart demonstrating a rotaxane almost a decade later [4]. Since then there have been many examples of topological molecules [1] and demonstrated applications include as nanometric electronic switches, liquid crystals and other new materials [5].
DNA origami is a DNA nanotechnology in which a long single-stranded DNA “scaffold” is shaped by the action of many short “staple” strands which bind to cognate sequences distributed throughout the scaffold [6, 7]. Typically, the scaffold strand is thousands of bases long, providing enough material for the construction of complex and rigid structures such as dynamic containers whose opening can be programmed in response to stimuli [8, 9]. While customization of scaffolds can be challenging, numerous examples have been reported including those that are shortened [10, 11], extended [12-14] or otherwise modified to provide arbitrary length and sequences [15-18].
DNA origami is a widely used DNA nanotechnology allowing construction of 2D and 3D nanometric shapes. The designability and rigidity of DNA origami makes it an ideal material for construction of topologically linked molecules such as catenanes which are attractive for their potential as motors and molecular machines (eg. US2013224859, WO15177666, US2017108517, CN106770049, CN107083389, CN107124178, CN107469088, WO18129333, US2020031663, KR102129784).
Efforts to connect together discrete DNA origami structures have included hybridisation of sticky ends [19] or base stacking [7, 20, 21, 22] and have achieved gigadalton scale structures [23] and fully addressable semi-micrometre scale tiles [24].
Topologically linked DNA molecules are known to occur in nature [25] and are also attractive as artificial constructs in DNA nanotechnology. In recent years catenated ssDNA rings have been designed and produced in vitro using a number of methods. Typically, these involve enzymatic ligation of short linear DNA components following geometric pre-arrangement by hybridisation of short complementary sequences [26-29] or conjugation with dsDNA-binding moieties [30]. The resulting structures have been shown capable of functioning as switches [26, 31] and rotary motors [27, 32]. However, this approach results in small (sub-200 nt) catenanes, essentially an order of magnitude smaller than required to make complex DNA origami structures. Production of considerably longer catenated ssDNA circles of a length suitable for DNA origami has been demonstrated in work which used Ena/Vasp-like protein and Topoisomerase 1 [33]. However, this approach showed poor efficiency, generated a range of catenated products, and has not been applied for DNA origami production.
Despite these challenges, topologically linked DNA origami remains an attractive goal due to its high molecular weight and resulting greater structural robustness compared to classical topologically linked molecules. As a result, it has the potential to construct molecular machines with increased functionality and sophistication due to the ability to irreversibly link together discrete DNA origami structures.
Very few examples of topologically linked DNA origamis have been demonstrated to date. The first that we are aware of was in 2010 when a 2-ring DNA origami catenane was produced [34]. This was achieved by splitting a DNA origami mobius strip followed by removal of selected staple strands. In this case, and crucially, the topology of the two connected scaffold strands is such that decatenation can be achieved without covalent bond breaking. More recently a second 2-ring DNA origami catenane was demonstrated. Here, each of the two rings was made from four individual DNA origamis which were joined together via additional staple strands with the help of a gold particle templated approach [35]. As with the first example, removal of noncovalently bound staple strands results in decatenation of the structure. In contrast a truly catenated scaffold can only be decatenated by covalent bond breaking and may be preferable for the extra stability it would provide to the linked DNA origami components.
Two-component DNA origami structures which could benefit from being produced as true catenanes include rotaxanes, the first of which was demonstrated in 2016 [36] in a structure where two discrete DNA origamis constituted the axle and ring. These were assembled first with the ring in an open form which was then partially wrapped around the axle before being mechanically “clamped” in place by complementary sequence extended from staple strands. More recently another rotaxane was produced from a single template DNA origami wherein the ring and the axle of the rotaxane precursor was connected via ssDNA regions which were then specifically removed by Cas12a to produce the final rotaxane [37].
A common pattern in all topologically linked DNA origami structures to date is that they are specifically constructed such that predetermined DNA origami subunits are positioned with respect to each other. This is followed by disconnection of the “linkers”—a subset of staples, base-pairing at specific sites as well as a part of the scaffold, resulting in a topologically linked product. A negative consequence of this approach is that the construction method is design specific. The final, folded DNA origami structures are required to be produced first, prior to the topological linkage being formed. This is in contrast to the strength of the original DNA origami concept whereby starting with a scaffold strand, almost any arbitrary structure can be designed and produced.
US2014141211 discloses methods for self-assembly of arbitrarily-shaped metal nanostructures using specifically-designed patterns on nucleic acid scaffolds.
US2017066796 discloses compositions and methods for engineering wireframe architectures and scaffolds of increasing complexity by creating gridiron-like DNA structures. A series of four-arm junctions are used as vertices within a network of double-helical DNA fragments. Deliberate distortion of the junctions from their most relaxed conformations ensures that a scaffold strand can traverse through individual vertices in multiple directions. DNA gridirons, ranging from two-dimensional arrays with reconfigurability to multilayer and three-dimensional structures and curved objects, can be assembled.
WO18218646 discloses a method for stepwise assembling multiple nucleic acid origami units into a nucleic acid nanostructure.
However, an alternative, universal method for production of topologically linked DNA origami is still needed.
The present invention is conceived to solve the aforementioned problem, and an object of the present invention is to provide a method of producing a topologically linked two-component DNA origami where the linkage is made at the level of two single-stranded scaffold strands and is a true catenane which cannot be separated except by covalent bond breakage.
The solution to the above-described technical problem is provided by subject invention which has been defined by enclosed claims.
In connection with the present invention, “topogami” is the term given to a form of DNA origami (Rothemund '06 Nature 10.1038/nature04586) which is not constructed from a single scaffold strand as is normal for DNA origami constructs, but two or more interlocked scaffold strand rings which are covalently catenated. (Sakai et al. '21 ACS Nanoscience Au 10.1021/acsnanoscienceau.1c00027).
According to subject invention the catenated single stranded DNA circles can be produced and used as a universal scaffold for the production of topologically linked (catenated) DNA origamis where the individual linked structures can be of any arbitrary design. Assembly of these topologically linked DNA origami structures is achieved via a simple one-pot annealing protocol.
Subject invention provides a method for preparation of a pair of topologically interlocked circular ssDNA scaffold rings using Tn3 resolvase. Catenating at this fundamental level has two noteworthy outcomes shown here for the first time. Firstly, the system is universal: as catenation does not depend on the formation of a specific DNA origami structure, the catenated ssDNA rings we produced can, in principle, be used to construct almost any two origami structures in a catenated form. Secondly, unlike topologically linked DNA origami structures to date, the topological linkage can only be undone by breaking a covalent bond (i.e. DNA backbone cleavage) resulting in high stability of the produced structures.
The present invention is further are illustrated by the following Examples, which in no way should be construed as further limiting.
Production of Catenated ssDNA Scaffold Strands
It has been designed pTopoScaf, a vector consisting of two scaffold domains connected by parallel recognition sequences (res) in a negatively supercoiled parent plasmid [38] which are processed by Tn3 resolvase, a serine recombinase [39-41] as outlined in
Nt.BspQI nickase sites have been integrated on opposite strands of the parent vector such that after Tn3 mediated recombination, each ring of the two-ring catenane would bear a single nickase site. This allows conversion of catenated product into ssDNA Nt.BspQI nicking and subsequent exonuclease digestion, while nicked non-catenated parent plasmid will be removed during by exonuclease digestion (
Production of catenated scaffolds was confirmed by agarose gel electrophoresis where ScaI/BamHI double digest of the Tn3 treated plasmid indicated the recombination event leading to formation of two smaller circles, each bearing one cutting site (for relative position of BamHI and ScaI cutting sites in the plasmid and catenane please refer to
Further confirmation was provided by AFM analysis which showed clear evidence of catenated product compared to unreacted controls (
Using the catenated scaffold, a topologically interlocked DNA origami complex, named “topogami”, has been obtained. Topogami consists of two discrete DNA origami structures which are catenated at the level of the scaffold strands. To demonstrate this, two rectangular single layer DNA origamis as the two linked structures (
Overall, the data clearly showed the successful demonstration of topologically linked DNA origami used to produce two discrete and covalently catenated DNA origami structures.
Plasmid pMA21 [45] containing two of parallel Tn3 res sites and Escherichia coli DH5 (F− λ− Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 (rK−, mK+) phoA supE44 thi-1 gyrA96 relA1) amplification host cell were a gift from Prof. Marshall Stark.
Plasmid pTopoScaf was derived from pMA21 by introducing a second Nt.BspQI nickase site (GCTCTTCN↓) by PCR using the following mutagenic primers:
Amplification was performed in a 20 μL reaction in 1×HF buffer (NEB), containing 50 ng template DNA, 1 μM of each primer, 500 μM dNTP mix and 1 μL Phusion polymerase. The PCR cycling program was 98° C. for 60 s followed by 20 cycles at 98° C. for 15 s; 55° C. for 15 s and 72° C. for 140 s and a final elongation step at 72° C. for 4 min. After completing the PCR reaction, methylated template DNA was removed by incubation with 1 μL FastDigest DpnI (Thermo Fisher Scientific) for 15 min at 37° C. An aliquot of 5 μL was transformed into chemo-competent E. coli DH5 and plated on Luria-Bertani plates containing 100 μg/ml ampicillin. The resulting mutant plasmid was verified by DNA sequencing.
Supercoiled plasmid DNA of pTopoScaf was purified from transformed E. coli strain DH5. Bacteria were grown over night in LB containing 100 μg/ml ampicillin. Plasmid was isolated using either the GenElute HP Select Plasmid Gigaprep Kit (Sigma Aldrich) or GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). DNA concentration was estimated from absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer. If required DNA was concentrated using EtOH precipitation.
Tn3 resolvase was a kind gift of Prof. Marshall Stark. Tn3 resolvase was diluted and stored in storage buffer (20 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 1 M NaCl, 50% v/v glycerol). Plasmid catenation was carried out as previously reported [41] with slight modifications. In brief, Tn3 resolvase was diluted 20-fold into 50 μL reaction buffer (10 mM MgCl2, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.2) containing 2.5 μg DNA. Reactions were carried out for 2 h at 37° C. The Tn3 catenation reaction was stopped by heating for 10 min at 70° C. After cooling to room temperature, 2 μL Nt.BspQI (NEB, 10,000 U/ml) were added and the reaction was incubated for 1 h at 50° C. followed by 20 min at 80° C. The nicked DNA was diluted with 50 μL 1×Exo III buffer (6.6 mM Tris-HCL pH 8, 0.66 mM MgCl2). 1 μL exonuclease III (Thermo Fisher Scientific, 200 U/μL) and 4 μL exonuclease I (Thermo Fisher Scientific, 10 U/μL) was added and incubated for 4 h at 37° C. followed by 10 min at 70° C. Incubations were carried out in 50 μL aliquots in a thermocycler with heated lid. Reactions were analysed by running 100 ng DNA for on a 1.2% agarose gel followed by subsequent staining with ethidium bromide and imaging by a UV transilluminator. To purify single stranded DNA from enzymes the products were purified by phenol:chloroform extraction, and concentrated 10 times by EtOH precipitation and resuspension in milli-Q water.
For design of DNA origami structures cadnano was used [46]
DNA origami structures were assembled in a one-pot reaction by mixing catenated scaffold and staple strands at final concentrations of 10 nM and 60 nM respectively. Folding buffer contained 5 mM Tris, 1 mM EDTA and 10 mM MgCl2. To assemble structures, DNA strands were incubated at 80° C. for 10 min followed by cooling from 79° C. to 25° C. with a decrease of 1° C. per 1 min in a thermocycler.
1.5 μL of 2 nM of assembled DNA origami sample was applied to freshly cleaved mica and incubated for 1 min followed by addition of 20 μL of folding buffer and immediately by 1.5 μL of 100 mM NiCl2. The specimen was measured in liquid by AFM (Dimension Icon, Bruker) working in PeakForce QNM® mode. ScanAsyst-Fluid+ probes (Bruker) with nominal spring constant equal to 0.7 N/m and sharpened tip necessary for high-resolution imaging in fluid (nominal radius equal to 2 nm) were used in all the measurements.
HS-AFM analysis was performed using a bespoke HS-AFM (NanoLSI, Kanazawa University) with a BL-AC10FS-A2 cantilever probe. DNA origami sample was loaded on freshly cleaved and nickel chloride treated mica and imaged in the folding buffer in tapping mode in liquid.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21461586.6 | Sep 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/075047 | 9/9/2022 | WO |
