DNA unfolding using free end tag flow modification reagents

Abstract

Methods are provided for utilizing the charged nature of DNA to facilitate DNA extension and manipulate its orientation. The method includes attaching highly charged molecules to the free ends of a DNA molecule to transform the DNA molecule into a pseudo-dipole. Upon application of an external electric field, charge separation can cause the DNA to extend and / or align with the electric field lines. This method provides a practical means of achieving a controlled, unfolded state of DNA with a predetermined orientation.

Description

Invention Field

[0001] In at least one aspect, this disclosure relates to a method for unfolding DNA. Background of the Invention Deciphering the genetic information encoded in DNA molecules requires the ability to induce and maintain transitions from the native folded structure to a more unfavorable extended state. Nanoscale confinement and electrophoretic forces hold promise for promoting DNA unfolding. However, effectively guiding DNA into confined spaces without generating excessive forces (which accelerate DNA and thus impair sensing and mapping applications) remains a challenge. A core issue lies in the limited ability to apply non-uniform forces along the DNA axis to promote stretching and unfolding. There is a need for methods to effectively promote DNA extension and manipulate its orientation. Invention Overview In various aspects, methods for manipulating the conformation and / or orientation of DNA molecules are provided. These methods include obtaining a DNA molecule; modifying the ends of the DNA molecule by coupling charged molecules to the ends of the DNA molecule; applying an external force to the DNA molecule; and adjusting the strength of the external force to induce a change in the conformation and / or orientation of the DNA molecule.

[0004] In other respects, methods for unfolding DNA molecules are provided. These methods include obtaining a DNA molecule; modifying the ends of the DNA molecule by coupling charged molecules to the ends of the DNA molecule; and subjecting the DNA molecule to an electric field.

[0005] In other respects, methods for orienting DNA molecules are provided. These methods include obtaining a DNA molecule; modifying the ends of the DNA molecule by coupling a polyionic tag to the ends of the DNA molecule to generate a pseudo-dipole state; subjecting the DNA molecule to an electric field; and adjusting the strength of the electric field to induce alignment of the DNA molecule with the axis of the electric field. Brief description of the attached diagram Figure 1A A single-stranded DNA molecule (ssDNA) with a drag tag is shown subjected to an electric field.

[0007] Figure 1B This shows the normalized end-to-end distance / maximum DNA length for ssDNA under different force intensities, derived from molecular dynamics simulations. <r> / Lmax.

[0008] Figure 1C A double-stranded DNA molecule (dsDNA) with a drag tag is shown subjected to an electric field.

[0009] Figure 1D This shows the normalized end-to-end distance / maximum DNA length for dsDNA under different force intensities, derived from molecular dynamics simulations. <r> / Lmax.

[0010] Figure 2A shows a double-stranded DNA molecule (dsDNA) with a drag tag subjected to an electric field.

[0011] Figure 2B illustrates the use of drag tags to obtain control over dsDNA orientation.

[0012] Figure 2C A single-stranded DNA molecule (ssDNA) with a drag tag is shown subjected to an electric field.

[0013] Figure 2D Drag tags are used to gain control over ssDNA orientation. Invention Details As requested, detailed embodiments of the invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely examples of the invention and may be embodied in various and alternative forms. The drawings are not necessarily drawn to scale; some features may be enlarged or reduced to show details of specific components. Therefore, the specific structural and functional details disclosed herein should not be construed as limiting, but rather serve only as a representative basis for teaching those skilled in the art to employ the invention in various ways.

[0015] Except as expressly indicated in the embodiments or when otherwise specified, all quantities indicating the amount of reaction and / or materials or conditions used in this specification shall be understood to be modified by the word "about". The initial definition of abbreviations or other acronyms applies to all subsequent uses of the same acronym herein, and with necessary modifications, applies to the conventional grammatical variations of the originally defined acronym; and, unless expressly stated otherwise, the measurement of properties shall be determined by the same technique as previously or subsequently referred to for the same property.

[0016] Unless otherwise stated, 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 pertains.

[0017] It should also be understood that this disclosure is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, change. Furthermore, the terminology used herein is for describing particular embodiments only and is not intended to be limiting in any way.

[0018] It must also be noted that, as used in the specification and appended claims, the singular forms "a," "an," and "the" contain plural indicators unless the context clearly indicates otherwise. For example, references to components in the singular are intended to include multiple components.

[0019] The terms "or" and "and" can be used interchangeably and can be understood as meaning "and / or".

[0020] The term "comprising" is synonymous with "including," "having," "containing," or "characterized in." These terms are inclusive and open-ended and do not exclude additional, unlisted elements or method steps.

[0021] The phrase "consisting of" excludes any element, step, or component not specified in the claim. When this phrase appears in the body clause of a claim, rather than immediately following the preamble, it only limits the element described in that clause; other elements are not excluded from the claim as a whole.

[0022] The phrase "consistent essentially of..." limits the scope of the claim to the specified materials or steps, plus those materials or steps that do not substantially affect the essential and novel characteristics of the subject matter for which protection is sought.

[0023] The terms "comprising," "consisting of," and "substantially composed of" may be used interchangeably. When one of these three terms is used, the subject matter disclosed herein and for which protection is sought may include the use of any of the other two terms.

[0024] The terms "complementarity," "complementary," or "complementary" can refer to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence through the traditional Watson-Crick or other non-traditional types.

[0025] When referring to DNA molecules, the phrase "conformational change" can be used to describe changes in the spatial configuration of DNA molecules, including bending or stretching of DNA molecules, or changes in their number of twists, coils, or connections.

[0026] When referring to the use of DNA, the terms "unfolding," "stretching," "extending," or "extension" can be used interchangeably to describe conformational changes in DNA molecules that cause the DNA molecule to tend toward a more linear conformation.

[0027] The terms "charged molecule", "tag" and "drag tag" may be used interchangeably throughout this disclosure.

[0028] The terms "highly charged" or "supercharged" may be used interchangeably throughout this disclosure and refer to molecules having a strength greater than that of DNA's negative linear charge density. The linear charge density of DNA refers to the amount of charge in a segment of DNA. The linear charge density of DNA can vary depending on the molar concentration of counterions. Counterions are positively charged ions to which DNA, along with water molecules, is exposed under physiological conditions. A supercharged or highly negatively charged molecule refers to a molecule having a linear charge density greater than or equal to -2e / molecule relative to the linear charge density of the DNA molecule to which it is coupled. A supercharged or highly positively charged molecule refers to a molecule having a linear charge density greater than or equal to 1e / molecule relative to the linear charge density of the DNA molecule to which it is attached. "e" refers to the elementary charge, which is the charge on a single proton or electron (1.602176487(40)×10⁻¹⁰). -19 coulomb).

[0029] Throughout this application, when references are made to publications, the disclosures of those publications, in their entirety, are incorporated herein by reference to provide a more comprehensive description of the prior art in the field to which this invention pertains.

[0030] DNA molecules contain the genetic information that controls cellular development in all living organisms. This crucial function forms the basis for the aspirations of personalized medicine and targeted therapies. Understanding the specific gene sequences and mutations that cause disease states can potentially be used to develop personalized therapeutics and also to answer fundamental questions in biology.

[0031] In its natural form, DNA undergoes significant conformational changes, including bending and twisting, resulting in tightly packed structures that can be accommodated within tiny cells or cell nuclei. This inherent folding of DNA presents challenges in accessing the encoded genetic information for mapping and sequencing purposes. Unfolding DNA is therefore crucial for the analysis of DNA sequences and structures, as well as the utilization of genetic information in biological and medical applications. Various techniques have been proposed for unfolding DNA molecules or manipulating their orientation, including hydrodynamics, optical tweezers, and nanoconfinement. These techniques vary in their level of success and complexity.

[0032] The key to DNA unfolding lies in the ability to apply non-uniform forces along the DNA strand, causing different segments of the molecule to shift relative to each other. Due to the uniform charge distribution of the molecule and the constant charge / friction ratio during free-solution electrophoresis, simply applying electrostatic forces to native DNA may not be sufficient. However, by chemically attaching charged end tags to the DNA molecule, a situation can be created where, under the influence of an external electric field, super-negatively or positively charged ends experience different forces (proportional to the sign and strength of the charge).

[0033] The concept of DNA mobility modifiers has been explored to achieve size-based DNA separation in free-solution electrophoresis. This approach involves using drag tags, which can be neutral or charged, to act as hydrodynamic parachutes, slowing down smaller DNA molecules compared to larger ones. By manipulating the chemistry at the DNA ends, it becomes possible to control DNA unfolding and orientation in a relatively simple and accessible manner.

[0034] In one or more embodiments provided herein, methods are disclosed for utilizing the charged properties of DNA to promote DNA elongation and manipulate its orientation. The method according to at least one embodiment involves attaching a highly charged molecule (positive or negative), referred to as a "tag" or "drag tag," to one of the free ends of a DNA molecule to convert the DNA molecule into a pseudo-dipole. Upon application of an external electric field, charge separation can induce DNA elongation and alignment with the electric field lines. This method provides a practical means of achieving a controlled, unfolded state of DNA with a predetermined orientation.

[0035] One or more implementations of the method may include chemically altering one or both ends of a DNA molecule to add a super-negatively or positively charged molecule. Applying an external electric field to the chemically altered DNA molecule can generate a non-uniform force along the DNA axis due to the generation of pseudo-dipole states. With appropriate chemical functionalization and electric field strength, DNA unfolding can be achieved in a relatively simple manner.

[0036] According to certain embodiments, chemically altering the ends of a DNA molecule can include chemically attaching a tag (e.g., a "drag tag") to the ends of the DNA molecule. Examples of suitable drag tags include, but are not limited to, polyanionic or polycationic tags. Polyanionic tags can include acrylamide copolymers, alginates, lignin sulfonates, pectin, polyacrylic acid and its copolymers, polyethylene sulfate, polycarboxylic acids, polysaccharides, or polystyrene sulfonic acid. Polycationic tags can include phenols, polyallylamine, poly(4-vinylbenzyltrimethylammonium) salts, polydiallyl dimethylammonium salts, polyethyleneimine, polyethyleneamine, polyvinylpyridine, polyvinylammonium salts, spermine or spermidine, peptide sequences (e.g., polylysine), amino plastics (e.g., melamine resins), or polyamide amines (dendritic polymers).

[0037] Depending on the application, drag tags can have linear, branched, or cross-linked morphologies. Dendritic polymers can be fine-tuned in terms of size, charge, and three-dimensional branching. This feature allows for fine-tuning of speed and alignment to regulate, for example, how DNA moves rapidly through a constrained space.

[0038] Drag tags can be further functionalized with various functional groups that allow coupling with modified or unmodified DNA. For example, drag tags can be modified with residues that allow for bioorthogonal click chemistry, such as alkynes, azides, trans-cyclooctene, tetrazines, tetrazolium, azirines, ketones, alkenes, oximes, protected alkylamines, or aldehydes; alkenes, alkynes, amides, or thiols; biotin or streptavidin; phosphoramids that allow for solid-phase synthesis, such as those binding to DNA; activated esters (succinimide esters) and amines; or aryl halides, borate esters, thiols, and alkenes for metal-catalyzed carbon-carbon bond formation.

[0039] The strategy of coupling a drag tag of interest to a DNA molecule, as mentioned above, can be used for direct coupling of the drag tag to DNA. Alternatively, the drag tag can be indirectly coupled to the DNA molecule via PCR, hybridization, or ligation of an oligonucleotide conjugated to the drag tag, which is the reverse complement of the DNA of interest. Depending on the application, these conjugates can act as primers or unique molecular identifiers (UMIs). In such cases, conjugation can be achieved via solid-phase synthesis, as an example. The conjugated oligonucleotide can be composed of native nucleic acids or modified nucleic acids. Modified nucleic acids can, for example, be used to increase the melting temperature of hybridization or to regulate the stiffness of the double strand. Suitable modified nucleic acids include, for example, phosphate-thiocyanate nucleic acids (PNAs), zwitterionic nucleic acids (ZNAs), locked nucleic acids (LNAs), etc.

[0040] In other embodiments, polyionic tags can be attached to DNA molecules by first introducing an initiator or chain transfer portion to the end of the DNA molecule via one of the aforementioned chemicals, followed by the addition of monomers to form a polymer chain.

[0041] In other embodiments, besides stretching the DNA molecule by adding drag tags to one or both ends, molecules that bind to internal sites on the DNA molecule can be added. For example, single-stranded binding proteins such as gp32 and recA can unwind single-stranded DNA. Like dendritic polymers, these fairly large protein tags can be used to control the number of DNA molecules simultaneously filling a constrained space. Furthermore, these proteins can guide DNA translocation through electrostatic fields, especially in constrained spaces. Additionally, these large tags can be used to fine-tune the speed at which DNA moves in an electrostatic field.

[0042] DNA molecules can be obtained by isolating from any cell, tissue, or organism, or generated, for example, from DNA or RNA molecules from any cell, tissue, or organism via PCR. DNA molecules can also be obtained from clinical or environmental samples, including, for example, skin swabs, bodily fluid samples, or swabs from environmental surfaces. Alternatively, DNA molecules can be synthesized, for example, through oligonucleotide synthesis. DNA molecules can be single-stranded or double-stranded and can include modified nucleotides. The length of DNA molecules can range from about 200 to about 500 base pairs.

[0043] DNA molecules to be manipulated can be delivered to the device. The device may have regions containing semi-solid or non-solid materials into which DNA molecules can be delivered and through which DNA molecules can move. The device may include at least two electrodes spaced apart from each other, with the semi-solid or non-solid material occupying the space between the electrodes. When activated, the electrodes can generate an electric field.

[0044] A controller can be used to implement the methods described herein. For example, the controller can drive the delivery of semi-solid or non-solid substances to a device, and drive the delivery of DNA molecules to a substance. The controller can also activate electrodes to generate an electric field.

[0045] The processes, methods, or algorithms disclosed herein may be deliverable to / implemented by a processing device, controller, or computer, which may include any existing programmable electronic control unit or dedicated electronic control unit. Similarly, processes, methods, or algorithms may be stored as data and instructions executable by a controller or computer in various forms, including but not limited to information permanently stored on non-writable storage media such as ROM devices, and information variablely stored on writable storage media such as floppy disks, magnetic tapes, CDs, RAM devices, and other magnetic and optical media. Processes, methods, or algorithms may also be implemented in an executable software object. Alternatively, processes, methods, or algorithms may be embodied, in whole or in part, using suitable hardware components such as application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), state machines, controllers, or other hardware components or devices, or a combination of hardware, software, and firmware components.

[0046] Example: Molecular dynamics simulations were used to evaluate the feasibility of a drag-tag strategy for DNA extension. The strengths of forces and external fields were assessed and correlated with the degree of DNA extension. The DNA molecules used in the simulations were approximately 250 base pairs long.

[0047] Figure 1A-1D The behavior of DNA molecules under stretchable forces is shown, derived from molecular dynamics simulations. A positive tag attached to a single free end of a DNA molecule is simulated without loss of generality. Figure 1A A tagged ssDNA molecule is shown. Figure 1B It shows information about DNA molecules, for example Figure 1A The simulation results of the DNA molecule shown. Figure 1C A tagged dsDNA molecule is shown. Figure 1D It shows information about DNA molecules, for example Figure 1C The simulation results of the DNA molecule shown. Overall, from Figure 1B and 1D The results of the simulations shown can be transferred to cases where the tag is used to generate two oppositely charged ends, or a supernegative drag tag is coupled at one end. The results can also be transferred to DNA molecules of approximately 200 to approximately 10,000 base pairs in length. Now refer to... Figure 1B and 1D Under the application of an external field, the overall length of a DNA molecule increases compared to the field-free state. This is observed by examining the average end-to-end distance of the DNA. <r>The evolution of is evaluated. To ensure the generality of the graph, <r>The strength of the DNA extension is normalized by the maximum DNA length (Lmax). The extent of DNA extension strongly depends on the properties of the DNA molecule (e.g., single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA)). Without external force, rigid dsDNA (characterized by a large continuous length Lp) is already ~70% of Lmax. An increase in the electric field (E) causes the DNA molecule to reach ~95% Lmax. The dragging tag strategy is more relevant for ssDNA, which is much more flexible than dsDNA and curls up to ~35% Lpmax in the absence of external force. Therefore, under the application of external force, a greater degree of extension is observed. <r>The basic increase. The strength of the force can be controlled in different ways, including by increasing the strength of the E field, increasing the charge (q) of the towed tag, or increasing the total number of molecules constituting the towed tag (through...). Figure 1B and 1D The difference in marker size is represented in the graph. Figure 1B and 1D The main plot highlights the feasibility of the disclosed drag tag modification design and the external operating conditions for achieving DNA unfolding.

[0048] In addition to achieving the unfolded state of the DNA molecule, the drag-tag strategy can also provide control over the orientation of the DNA molecule. This is due to the separation of charges, resulting in the DNA molecule being in a pseudo-dipole state. Thermodynamically, the formed dipole will wave, with its long axis aligned with the E-field line and its more positively charged end pointing towards the field direction. Figure 2A shows a tagged dsDNA molecule. Figure 2B shows the results from simulations using DNA molecules such as those shown in Figure 2A. Figure 2C A tagged ssDNA molecule is shown. Figure 2D It shows the results from DNA molecules, for example Figure 2C The results of simulations of the DNA molecule shown are illustrated. Figures 2B and 2D show the probability of the DNA axis aligning with the field axis (within any angle of 30°). This probability is altered by subjecting the DNA molecule to an external force. Adjusting the strength of the force applied to the DNA molecule can change this probability. For example, increasing the force can achieve a probability close to 1. The force can be increased by increasing the strength of the E field, increasing the charge (q) of the drag tag, or increasing the total number of molecules constituting the drag tag. The force can also be decreased by decreasing these parameters. However, the effectiveness of the dragging strategy in achieving a specific orientation depends on the properties of the DNA molecule. Figure 1B and 1D The results in the previous study showed that dsDNA, due to its rigidity and low volatility, could achieve better alignment with the field line. On the other hand, highly volatile ssDNA required significantly greater force to achieve alignment. This is achieved through… Figure 2D The low probability of small markers (a small number of tagged molecules) is obvious.

[0049] While exemplary embodiments have been described above, they are not intended to describe all possible forms of the invention. Rather, the language used in the specification is descriptive rather than restrictive, and it should be understood that various changes can be made without departing from the spirit and scope of the invention. Furthermore, features of the various embodiments can be combined to form further embodiments of the invention.< / r> < / r> < / r> < / r> < / r>

Claims

1. A method for manipulating the conformation and / or orientation of a DNA molecule, the method comprising: Obtain DNA molecules; The ends of DNA molecules are modified by coupling charged molecules to the ends of DNA molecules. Apply an external force to the DNA molecule; and The intensity of the external force is adjusted to induce the DNA molecule to change its conformation and / or orientation.

2. The method of claim 1, wherein applying an external force to the DNA molecule comprises subjecting the DNA molecule to an electric field.

3. The method according to claim 2, wherein adjusting the strength of the external force includes adjusting the strength of the electric field.

4. The method of claim 2, wherein adjusting the intensity of the external force includes adjusting the charge of the charged molecules.

5. The method of claim 1, wherein the charged molecule is a polyanionic tag.

6. The method of claim 1, wherein the charged molecule is a polycationic tag.

7. The method of claim 1, wherein charged molecules are coupled to each end of the DNA molecule.

8. The method of claim 1, further comprising coupling the charged molecule to the end of the DNA molecule via PCR, hybridization, or ligation of an oligonucleotide conjugated to the charged molecule, wherein the oligonucleotide is the reverse complement of the DNA molecule.

9. A method for unfolding a DNA molecule, comprising: Obtain DNA molecules; The ends of DNA molecules are modified by coupling charged molecules to the ends of DNA molecules. and The DNA molecule is subjected to an electric field.

10. The method of claim 9, wherein the charged molecule is a polyanionic tag.

11. The method of claim 9, wherein the charged molecule is a polycationic tag.

12. The method of claim 9, wherein charged molecules are coupled to each end of the DNA molecule.

13. The method of claim 9, wherein the DNA molecule is single-stranded.

14. A method for orienting DNA molecules, comprising: Obtain DNA molecules; The ends of DNA molecules are modified by coupling polyionic tags to the ends of DNA molecules to generate pseudo-dipole states. The DNA molecule is subjected to an electric field; and The intensity of the electric field is adjusted to induce the DNA molecule to align with the axis of the electric field.

15. The method of claim 14, further comprising adjusting the charge of the polyionic tag to induce the DNA molecule to align with the axis of the electric field.

16. The method of claim 14, further comprising adjusting the total number of molecules constituting the polyionic tag to induce the DNA molecules to align with the axis of the electric field.

17. The method of claim 14, wherein the DNA is single-stranded.

18. The method of claim 14, wherein the polyionic tag is a polyanionic tag.

19. The method of claim 18, wherein the polyionic tag is a dendritic polymer.

20. The method of claim 14, further comprising coupling the polyionic tag to the end of the DNA molecule via PCR, hybridization, or ligation of an oligonucleotide conjugated to the polyionic tag, wherein the oligonucleotide is an inverse complement of the DNA molecule.

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