WO2024220771A1

WO2024220771A1 - Rapid enzyme-free release of single-stranded dna from unmodified dsdna templates

Info

Publication number: WO2024220771A1
Application number: PCT/US2024/025356
Authority: WO
Inventors: Wolfgang Pfeifer; Carlos Castro; Enrique Ruiz; Patrick HALLEY
Original assignee: Ohio State Innovation Foundation
Current assignee: Ohio State Innovation Foundation
Priority date: 2023-04-21
Filing date: 2024-04-19
Publication date: 2024-10-24
Anticipated expiration: 2025-10-21

Abstract

Disclosed herein is a rapid way to prepare ssDNA from various double-stranded (ds) DNA sources. The method is based on competitive binding of short, single-stranded DNA oligonucleotides (short, complementary oligonucleotides (SCOs)), which are identical to the target sequence to be released. Short single-stranded DNA oligonucleotides are commercially available and can be synthesized with arbitrary sequence, such that a complete set of strands that are piecewise complementary to any dsDNA target sequence of interest can be easily prepared and ordered from a variety of commercial vendors. In this approach a set of strands are designed that are complementary to the opposing strand in the dsDNA template so competitive binding will release the ssDNA strand of interest.

Description

RAPID ENZYME-FREE RELEASE OF SINGLE-STRANDED DNA FROM UNMODIFIED DSDNA TEMPLATES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63/497,532, filed April 21 , 2023, and U.S. Provisional Application No. 63/552,823, filed February 13, 2024, which are hereby incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant Nos. 1933344 and 1921881 awarded by the National Science Foundation. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] It is costly and time consuming to produce single stranded DNA (ssDNA). It also generally involves hazardous chemicals, such as acrylamide.

SUMMARY OF THE INVENTION

[0004] Disclosed herein is a rapid way to prepare ssDNA from various double-stranded (ds) DNA sources. The method is based on competitive binding of short, single-stranded DNA oligonucleotides (short, complementary oligonucleotides (SCOs)), which are identical to the target sequence to be released. Short single-stranded DNA oligonucleotides are commercially available and can be synthesized with arbitrary sequence, such that a complete set of strands that are piecewise complementary to any dsDNA target sequence of interest can be easily prepared and ordered from a variety of commercial vendors. In this approach a set of strands are designed that are complementary to the opposing strand in the dsDNA template so competitive binding will release the ssDNA strand of interest.

[0005] A temperature-controlled cascade reaction is used to release the target ssDNA from dsDNA. First, all species of dsDNA into ssDNA are melted by incubating sample at 98°C for about 70 seconds. The temperature of the reaction is then lowered to 65-85°C for about 5 minutes. This allows the SCOs to hybridize to one of the long strands, preventing its reannealing. The key to having SCOs bind preferentially is using a certain length in terms of bases. The length of SCOs determines their melting temperature. The reaction temperature is lower than the melting temperature of SCOs, leading to SCOs binding to the long piece of DNA.

[0006] By including a suitable restriction enzyme, circular or supercoiled dsDNA can also be used as starting material. In this case, the temperature-controlled cascade reaction starts with the working temperature of the restriction enzyme, e.g. 37°C. Finally, the temperature of the reaction is reduced to 4°C and the freshly prepared DNA is purified and stored.

[0007] Single-stranded DNA is needed for many different applications, ranging from biophysics over gene delivery to DNA nanotechnology. In some embodiments, the method is used to prepare scaffolded DNA origami. Since scaffolded DNA origami is a sequence specific approach, formation of target structures indicates successful release of the target sequence.

[0008] Double-stranded DNA can be manufactured easier than single-stranded DNA. Removing one strand from a dsDNA can be done, using expensive instruments such as Streptavidin coated magnetic beads, using biotinylated primers during PCR in the production of the dsDNA. Alternatively, hazardous chemicals such as acrylamide are required during PCR reactions, performing Methanol responsive polymer PCR. Additionally, the disclosed approach can be performed with standard laboratory instruments and does not require new equipment. Finally, the disclosed method works with circular or supercoiled DNA.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF FIGURES

[0010] FIGs. 1A and 1 B show release of a 769 nt long ssDNA from a 769 bp linear PCR product using the method disclosed herein. Figure 1A is a schematic of the single-stranded release reaction, (i) double-stranded DNA is heated, (ii) separating the two individual

[0011] strands. During cooling, blocking strands competitively bind to one isolated strand, (iii) displacing the complementary strand. Different fluorophore labels were used to trace the respective species during agarose gel electrophoresis (FIG. 1 B), showing the release of single-stranded DNA. controls were enzymatically prepared in the agarose gel to compare reaction products to known species.

[0012] FIG. 2 shows release of a 769 nt long ssDNA from a 769 bp linear PCR product using the method disclosed herein. Control 1 contains double-stranded DNA of the given length. Control 2 contains single-stranded DNA of the given length.1x-25x contains different excess of blocking strands and 769 nt long dsDNA. These samples went through our temperature program and show single-stranded DNA. This agarose gel has been stained with Ethidium bromide to show all DNA present.

[0013] FIG. 3 shows no release occurs at room temperature (769 nt long ssDNA from a 769 bp linear PCR product). Control 1 contains a known standard (1 kb ladder from New England Biolabs). Control 2 contains single-stranded DNA of the given length. Release 1-4 contains double-stranded DNA and short, complementary oligonucleotides. Release 1 uses the same concentration of short, complementary overhangs as double-stranded DNA. Release 2 uses the twice the concentration of short, complementary overhangs as double-stranded DNA. Release 3 uses the five times the concentration of short, complementary overhangs as doublestranded DNA. Release 4 uses the ten times the concentration of short, complementary overhangs as double-stranded DNA. The temperature protocol was NOT used for any sample. No signal corresponding to single-stranded DNA was observed, indicating that no release happens at room-temperature, allowing us to control the system through precise adjustments of the temperature.

[0014] FIG. 4 shows release of a 2118 nt long ssDNA from a 2118 bp linear PCR product. Control 1 contains double-stranded DNA of the given length. Control 2 contains singlestranded DNA of the given length. The release contains double-stranded DNA of 2118 bps, as well short, complementary oligonucleotides. This sample went through the temperature program and showed single-stranded DNA. This agarose gel has been stained with Ethidium bromide to show all DNA present.

[0015] FIG. 5 shows release of a 5386 nt long ssDNA from a circular phiX viral dsDNA. Control 1 contains a known standard (1 kb ladder from New England Biolabs). Control 2 contains double-stranded DNA of the given length. Control 3 contains single-stranded DNA of the given length. The release contains double-stranded DNA of 5386 bps, as well short, complementary oligonucleotides. This sample went through our temperature program and shows singlestranded DNA. This agarose gel has been stained with Ethidium bromide to show all DNA present.

[0016] FIGs. 6A to 6C shows realization of a DNA origami nanostructure from pUC19 (a circular, double-stranded expression vector), grown in E. coli. The double-stranded DNA is first enzymatically cleaved to yield linear double-stranded DNA. It is then used with our approach to release single-stranded DNA. Finally, the single stranded DNA is used to fold DNA origami nanostructures. FIG. 6A shows a coarse-grained simulation of a DNA origami nanostructure. FIG. 6B shows a full AFM image, displaying multiple DNA origami nanostructures. FIG. 6C shows a gallery of individual DNA origami nanostructures, clearly displaying the designed features, indicating that the full sequence of pUC19 is used to form these structures.

[0017] FIG. 7A shows release of single-stranded DNA from linear, double-stranded DNA. FIG. 7B shows subsequent folding of a DNA origami from the released single-stranded DNA. [0018] FIGs. 8A-8C show one-pot folding of DNA origami from dsDNA folding from linear dsDNA through thermal release of ssDNA with subsequent DNA origami (FIG. 8A), folding from supercoiled dsDNA through restriction of the template DNA, followed by the thermal release of ssDNA with subsequent folding of dsDNA (FIG. 8B), or release and folding with a 15 kb A DNA fragment or multiple shorter A DNA fragments (FIG. 80). Models not to scale.

DETAILED DESCRIPTION

[0019] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0020] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0022] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0023] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0024] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0025] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0026] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0027] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0028] In some embodiments, the first step is to create blocking strands from the entire target sequence starting at basel and ending at the last. Blocking strands are preferably designed to have similar length, which equals to similar melting temperature. Since not every dsDNA template’s lengths is a multiple of 60, one can adjust this slightly, e.g., the 769 dsDNA template used 2x60nt & 11x59nt; the 2118 dsDNA template used 3x46nt & 33x60nt. Release was obtained from the 769 dsDNA template with the blocking strand lengths of about 30, 60, and 90 nucleotides.

[0029] All blocking strands can then be combined into one solution of equal concentration. Double stranded template and corresponding blocking strands can then be combined with blocking strands in excess. 10 and 15 nM dsDNA template were used for the release experiments, usually with a 10x excess of blocking strands. A thermal protocol is then applied.

Embodiments

[0030] Embodiment 1 . A method for producing single stranded DNA (ssDNA) from a double stranded DNA (dsDNA) template, comprising

(a) providing ssDNA oligonucleotide blocking strands 30-90 nucleotides in length complementary to at least 50% of the length of the dsDNA template;

(b) mixing the blocking strands with the template with the blocking strands being at an equal or higher concentration than the template;

(c) incubating the mixture at a melting temperature above the melting temperature of the template for at least 60 seconds;

(d) incubating the mixture at an annealing temperature above the melting temperature of the template but lower than the melting temperature of the blocking strands for at least 5 minutes to allow the blocking strands to hybridize to the melted template, thereby preventing reannealing of the template and releasing a ssDNA template strand;

(e) lowering the temperature to a storage temperature; and

(f) isolating the ssDNA template from the mixture.

[0031] Embodiment 2. The method of embodiment 1 , wherein the template strand is a circular or supercoiled dsDNA, wherein step (b) further comprises adding a restriction enzyme to the mixture and incubating the mixture at the working temperature of the enzyme for at least 30 minutes.

[0032] Embodiment 3. The method of embodiment 1 or 2, wherein the ssDNA selfassembles into a DNA origami structure upon release.

[0033] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the melting temperature is about 98°C.

[0034] Embodiment 5. The method of any one of embodiments 1 to 4, wherein step (c) comprises incubating the mixture at a melting temperature above the melting temperature of the template for about 70 seconds.

[0035] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the annealing temperature is about 65-85°C.

[0036] Embodiment 7. The method of any one of embodiments 1 to 6, wherein step (d) comprises incubating the mixture at an annealing temperature above the melting temperature of the template for about 5 minutes. [0037] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the storage temperature is about 4°C.

[0038] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1 :

[0039] The production of single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA) templates is central to gene delivery, medicine, biotechnology, DNA nanotechnology and more. Research in these areas is often hindered due to the limitation in current methods of single-stranded DNA production, which can be costly, time-consuming, and lack ease-of-use and sequence variety.

[0040] Solid-phase synthesis struggles to achieve high yields for ssDNA larger than 200 bases, often containing many side products with increasing length. Enzymatic conversions of dsDNA to ssDNA can be expensive and require several intensive hours to complete. Hence there is a need for simple and versatile methods for ssDNA production. Presented here is a method for the quick release of target ssDNA strands from dsDNA, with lengths shown between 769 and around 15 kilobases. Target ssDNA were successfully isolated from linear, circular, and supercoiled templates, respectively. By using short, complementary oligonucleotides, we competitively isolate ssDNA from dsDNA templates (Fig. 1 A), which is visualized via agarose gel electrophoresis and used in subsequent folding of DNA origami nanostructures. A set of fluorophore-labeled oligonucleotides were used to specifically trace all species present in the sample, allowing one to easily identify reaction pathways and outcomes (Fig. 1 B). This method can be useful to applications in multiple fields, due to its straightforward nature, and ability to isolate ssDNA from a versatile range of dsDNA templates without the need to chemically modify the template. Furthermore, this approach relies solely on basic laboratory equipment, which makes adaptation easy and straightforward.

Example 2:

[0041] Scaffolded DNA origami relies on the designed and programmed interaction between many different single-stranded DNAs (ssDNA). While short oligonucleotides can be readily produced through solid-phase synthesis, production of the scaffold strand is mostly limited to a few available sources. Even though there has been significant effort to expand the available sources for long ssDNAs, sources of ssDNA are still significantly limited compared to widely available sources (e.g. plasmids) of double-stranded DNA (dsDNA). To address challenges related to source DNA materials for DNA origami fabrication, we have developed a new approache to fold DNA origami nanostructures using dsDNA sources of various size and complexity, ranging from linear duplexes to supercoiled plasmids.

[0042] The disclosed methods were validated using unique shapes with clear features for identification, and by probing the dynamical properties of the target structures. The first method used the competitive binding of oligonucleotides (‘blocking strands’) towards one strand to prevent reannealing of the latter with its complementary strand (Fig. 7A), thus, effectively releasing a long piece of ssDNA which can be used in DNA origami folding reactions. This led to successful folding of DNA origami nanostructures from dsDNA templates, ranging from around 700 bps to over 15,000 bps, but can be expanded to realize structures larger than 10 kbps.

[0043] These approaches increase the versatility of DNA origami, which we envision can provide significant advantages, for example, using commonly available dsDNA sources when scaling up folding for a variety of applications or leveraging existing plasmid sources with other beneficial features like protein or enzyme recognition sites.

Example 3:

[0044] While folding from linear dsDNA is already a significant improvement, increasing the library of potential scaffolds used in DNA nanotechnology, enabling circular and even supercoiled dsDNA will further leverage these options. We developed a simple, one pot reaction to rapidly release ssDNA in situ and subsequently fold it into DNA origami nanostructures. We show successful realization of DNA origami structures, folded from linear dsDNA, pUC 19 and X 174 plasmids, and A DNA (FIGs. 8A to 80). Insets show average over an oxDNA simulation trajectory Scale bar 100 nm).

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0046] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1 . A method for producing single stranded DNA (ssDNA) from a double stranded DNA (dsDNA) template, comprising

(e) lowering the temperature to a storage temperature; and

(f) isolating the ssDNA template from the mixture.

2. The method of claim 1 , wherein the template strand is a circular or supercoiled dsDNA, wherein step (b) further comprises adding a restriction enzyme to the mixture and incubating the mixture at the working temperature of the enzyme for at least 30 minutes.

3. The method of claim 1 , wherein the ssDNA self-assembles into a DNA origami structure upon release.

4. The method of claim 1 , wherein the melting temperature is about 98°C.

5. The method of claim 1 , wherein step (c) comprises incubating the mixture at a melting temperature above the melting temperature of the template for about 70 seconds.

6. The method of claim 1 , wherein the annealing temperature is about 65-85°C.

7. The method of claim 1 , wherein step (d) comprises incubating the mixture at an annealing temperature above the melting temperature of the template for about 5 minutes.

8. The method of claim 1 , wherein the storage temperature is about 4°C.