Method for screening compounds

Abstract

Disclosed herein are methods of screening candidate binding compounds for a plurality of target nucleic acids having the same sequence or different sequences using a device configured to detect changes in folding force and unfolding force of the target nucleic acids when bound to various candidate binding compounds. In the presence of a plurality of nucleic acid sequences, the methods described herein can also rapidly determine the specificity of small molecules or polypeptides for nucleic acid structures. Further, disclosed herein are methods of measuring binding kinetics of a binding compound to a target nucleic acid using a device configured to detect changes in folding force and unfolding force of the target nucleic acid when bound to candidate binding compounds of different concentrations.

Description

[0001] Cross-referenced applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 326,370, filed on April 1, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporation by reference into the sequence listing

[0004] This application contains a sequence listing, which has been submitted through the Patent Center. The name of the sequence listing is 201326.701601_PCT_SL.xml, created on March 31, 2023, and has a size of 43,892 bytes, and is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to methods and kits for identifying candidates capable of interacting with nucleic acids, proteins, or analogs thereof. Background Art

[0006] Candidates that can bind to a target molecule can be identified by high-throughput screening. High-throughput screening allows the use of automated equipment to test thousands to millions of molecules. To identify candidates, a strategy consists of first identifying high-quality hits and then affine the selection, for example, by modifying the high-quality hits to design candidates. In parallel, various doses of selected candidates can also be tested to quantitatively analyze the behavior of the molecules and potentially predict their activity.

[0007] In order to improve the selection of these potential candidates and / or obtain more meaningful results, several methods have been developed, such as the very sensitive optical method "surface plasmon resonance (SPR)". SPR occurs when polarized light hits a conductive surface at the interface between two media. This produces an electronic charge density wave called a plasmon, which reduces the intensity of the reflected light at a specific angle (called the resonance angle) in proportion to the mass on the sensor surface. Therefore, this technology allows real-time detection of molecular interactions. However, this still relies only on binding between molecules. Attempts have been made to identify methods for improving the accuracy of selecting candidates, but the methods used are still challenging. Therefore, there is still a need for devices and methods for faster, more selective and accurate detection of new candidates acting on nucleic acids or proteins. Summary of the invention

[0008] Disclosed herein are methods for real-time screening or identification of binding molecules to the conformational structure of nucleic acid molecules.

[0009] In some embodiments, the method comprises: (a) connecting a plurality of nucleic acid molecules to a plurality of beads via one end of the nucleic acid molecule; (b) connecting a plurality of nucleic acid molecules to a plurality of features of a device via a second end of the nucleic acid molecule; (c) contacting a chamber with a binding molecule library, wherein each member of the binding molecule library is in contact with a plurality of single nucleic acid molecules in a single chamber; (d) approaching a magnet to move a plurality of beads along an axis of the chamber relative to a bottom surface, and measuring changes in the positions of the beads through a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (f) selecting a member of the binding molecule library that produces an unfolding force of the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (g) optionally, testing the identified binding molecule for activity as a medicine. In some embodiments, the plurality of features comprises at least 10,000 features. In some embodiments, the sensor of (d) comprises a camera CMOS capable of collecting photons reflected from the beads. In some embodiments, the sensor of (d) includes a CMOS capable of measuring the impedance caused by the up and down movement of the beads from the features. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is a DNA / RNA hybrid molecule. In some embodiments, a plurality of nucleic acid molecules are identical nucleic acid molecules. In some embodiments, a plurality of nucleic acid molecules are different nucleic acid molecules. In some embodiments, the force of the magnet relative to the beads is constant. In some embodiments, the force of the magnet relative to the beads is increased. In some embodiments, the nucleic acid molecule is about 20bp to about 350bp. In some embodiments, the nucleic acid molecule is greater than 20bp and at most greater than 1.5kb. In some embodiments, the nucleic acid molecule is about 20 bases to about 350 bases. In some embodiments, the nucleic acid molecule is greater than 20 bases and at most greater than 1.5 bases. In some embodiments, the nucleic acid molecule is chemically synthesized. In some embodiments, the nucleic acid molecule is produced by in vitro transcription. In some embodiments, the nucleic acid molecule is produced by polymerase chain reaction. In some embodiments, the nucleic acid molecule is purified from at least one cell and can be compared with the nucleic acid from a diseased (cancer) cell. In some embodiments, the binding molecule is a small molecule. In some embodiments, the binding molecule is a polypeptide. In some embodiments, the device is configured to solve the difference in unfolding power of about 0.1 times to about 30 times between the members of the binding molecule library. In some embodiments, the connection of (a) includes biotin-streptavidin binding, wherein biotin is conjugated to one end of the nucleic acid, and streptavidin is conjugated to a bead.In some embodiments, the linking of (b) comprises a DNA oligonucleotide covalently linked to the base of each feature, wherein the DNA oligonucleotide has complementarity to the second end of the nucleic acid molecule.

[0010] In some embodiments, the method further comprises the step of contacting the nucleic acid with a small molecule, protein (such as an enzyme) that interacts with the nucleic acid and analyzing their binding. In some embodiments, the nucleic acid comprises (or is or consists of) an aptamer.

[0011] Also disclosed herein is a method for identifying a chemical labeling of an optimal binder in a binding molecule library of nucleic acid molecules. In some embodiments, the method includes: (a) connecting a plurality of nucleic acid molecules to a plurality of beads by a first end of a nucleic acid molecule; (b) connecting a plurality of nucleic acid molecules to a plurality of features of a device by a second end of a nucleic acid molecule; (c) contacting a chamber with a binding molecule library, wherein each member of the binding molecule library is in contact with a single nucleic acid molecule; (d) approaching a magnet to move a plurality of beads relative to the bottom surface along the axis of the chamber, and measuring the change in the position of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of a nucleic acid molecule in the presence of each member of the binding molecule library; (f) selecting a member of the binding molecule library that produces an unfolding force of a nucleic acid molecule, the unfolding force being greater than or less than the unfolding force of a nucleic acid molecule in the absence of a binding molecule; and (g) modeling selected members of the binding molecule library to identify a chemical labeling of an optimal binder in a binding molecule library. In some embodiments, a plurality of features include at least 10,000 features. In some embodiments, the sensor of (d) includes a camera CMOS capable of collecting photons reflected from beads. In some embodiments, the sensor of (d) includes a CMOS capable of measuring the impedance caused by the up and down movement of beads from features. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is a DNA / RNA hybrid molecule. In some embodiments, a plurality of nucleic acid molecules are identical nucleic acid molecules. In some embodiments, a plurality of nucleic acid molecules are different nucleic acid molecules. In some embodiments, the force of the magnet relative to the beads is constant. In some embodiments, the force of the magnet relative to the beads is increased. In some embodiments, the nucleic acid molecule is about 20bp to about 350bp. In some embodiments, the nucleic acid molecule is about 20 bases to about 350 bases. In some embodiments, the nucleic acid molecule is chemically synthesized. In some embodiments, the nucleic acid molecule is produced by in vitro transcription. In some embodiments, the nucleic acid molecule is produced by polymerase chain reaction. In some embodiments, the nucleic acid molecule is purified from cells. In some embodiments, the binding molecule is a small molecule. In some embodiments, the binding molecule is a polypeptide. In some embodiments, the device is configured to resolve differences in unfolding power of about 0.1 times to about 30 times between members of the binding molecule library. In some embodiments, the connection of (a) includes biotin-streptavidin binding, wherein biotin is conjugated to one end of the nucleic acid, and streptavidin is conjugated to the beads. In some embodiments, the connection of (b) includes a DNA oligonucleotide covalently connected to the bottom of each feature, wherein the DNA oligonucleotide has complementarity with the second end of the nucleic acid molecule.

[0012] Also disclosed herein is a method for determining the binding kinetics of a binding molecule to a nucleic acid molecule. In some embodiments, the method includes: (a) connecting a plurality of nucleic acid molecules to a plurality of beads by a first end of a nucleic acid molecule; (b) connecting a plurality of nucleic acid molecules to a plurality of features of a device by a second end of a nucleic acid molecule; (c) contacting a chamber with a binding molecule of increasing concentration; (d) applying a magnetic field sufficient to move each bead in a plurality of beads relative to the bottom surface of the chamber, and measuring the change in the position of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule according to the concentration change of the binding molecule; and (f) calculating the binding kinetics of the binding molecule to the nucleic acid molecule based on the change in the unfolding force according to the amount change of the binding molecule. In some embodiments, the unfolding force of the nucleic acid molecule according to the concentration change of the binding molecule is calculated for each concentration of the binding molecule at the same time. In some embodiments, the binding molecule has a dissociation constant (KD) of about 0.01nM to about 100mM.

[0013] Also disclosed herein is a method for determining the binding kinetics of a binding molecule and a nucleic acid molecule. In some embodiments, the method includes: (a) contacting the nucleic acid molecule connected to the fixed end of the chamber and the magnetic beads on the opposite end with the binding molecule of increasing concentration; and (b) determining the unfolding force of the nucleic acid according to the concentration change of the binding molecule, wherein the nucleic acid molecule is selected from the group consisting of HIV-1TAR, preQ1 riboswitch, pre-miR-21, MYC, APOC3, DM1, C9orf72, MAPT and FMR1. In some embodiments, the binding molecule is a small chemical molecule. In some embodiments, the binding molecule is a polypeptide. In some embodiments, the binding molecule has a dissociation constant (KD) of about 0.01nM to about 100mM. In some embodiments, the chamber is arranged in a device configured to measure the displacement of the beads of the nucleic acid according to the concentration change of the binding molecule. In some embodiments, the device includes multiple features. In some embodiments, multiple nucleic acid molecules are anchored to the bottom of multiple features, wherein each feature of the chamber includes a single nucleic acid molecule sequence in multiple nucleic acid molecule sequences. In some embodiments, a single concentration of binding molecules is added to the chamber, wherein the unfolding force of the nucleic acid according to the concentration variation of the binding molecules is calculated for each concentration of binding molecules. In some embodiments, the plurality of features comprises at least 10,000 features. In some embodiments, the nucleic acid molecule is HIV-1TAR. In some embodiments, the nucleic acid molecule is a preQ1 riboswitch. In some embodiments, relative to the wild-type preQ1 riboswitch, the preQ1 riboswitch comprises a mutation selected from the group consisting of: C15U, U7C, A10G and U6C. In some embodiments, the nucleic acid molecule is pre-miR-21. In some embodiments, the nucleic acid molecule is DM1.

[0014] In some embodiments, the nucleic acid molecule is a viral nucleic acid, a nucleic acid isolated from bacteria, preferably a nucleic acid isolated from a pathogenic viral agent or a pathogenic bacterial agent. In some embodiments, the nucleic acid is from a cell isolated from a cancer, or from a cell under pathological stress. In some embodiments, the nucleic acid molecule is a viral RNA isolated from an RNA virus. In some embodiments, the viral RNA is from a coronavirus. In some embodiments, the viral RNA is from a SARS virus, a MERS coronavirus, or a SARS-COV-2 virus.

[0015] Also disclosed herein are systems for practicing the methods disclosed herein; kits and containers comprising the systems disclosed herein; and devices comprising the systems or devices disclosed herein. In some embodiments, the present invention also provides systems or devices for real-time selection of molecules that bind to conformational states of nucleic acids.

[0016] Also disclosed herein is a system for screening binding molecules of nucleic acid molecules. In some embodiments, the system includes: (a) a plurality of nucleic acid molecules connected to a plurality of beads by one end of the nucleic acid molecule; (b) a plurality of nucleic acid molecules connected to a plurality of features of a device by a second end of the nucleic acid molecule; (c) a chamber having a binding molecule library, wherein each member of the binding molecule library is in contact with a plurality of single nucleic acid molecules in a single chamber; (d) a magnet that moves a plurality of beads along the axis of the chamber relative to the bottom surface; and (e) a sensor that measures changes in the bead position of the device based on the movement of the beads relative to the bottom surface.

[0017] Also disclosed herein is a system for determining the binding kinetics of a binding molecule to a nucleic acid molecule. In some embodiments, the system includes: (a) a plurality of nucleic acid molecules connected to a plurality of beads by one end of the nucleic acid molecule; (b) a plurality of nucleic acid molecules connected to a plurality of features of a device by a second end of the nucleic acid molecule; (c) a chamber having a binding molecule library, wherein each member of the binding molecule library is in contact with a plurality of single nucleic acid molecules in a single chamber; (d) a magnet that moves a plurality of beads along the axis of the chamber relative to the bottom surface; and (e) a sensor that measures changes in the bead position of the device based on the movement of the beads relative to the bottom surface.

[0018] Also disclosed herein are kits comprising: (a) one or more identical or different nucleic acid molecules comprising a directional sequence at one end and a biotin moiety at the other end; (b) a DNA handle comprising a first reactive group, wherein the DNA handle is capable of hybridizing with the directional sequence; (c) a feature of a device comprising a second reactive group capable of chemically reacting with the first reactive group; and (d) one or more beads comprising a streptavidin moiety capable of binding to the biotin moiety. In some embodiments, the kit further comprises a binding molecule library. In some embodiments, the binding molecule library comprises a small molecule library, a protein library, an antibody library, an aptamer library, and advantageously comprises a library of binding molecules. In some embodiments, the kit further comprises a buffer comprising a divalent cation in the range of 1 μM to 1 mM. In some embodiments, the divalent cation comprises Mg. 2+ , Mn 2+ 、Zn 2+ , Fe 2+ , Cu 2+ or Cd 2+ In some embodiments, the chemical reaction is an esterification reaction. In some embodiments, the chemical reaction is click chemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the exemplary embodiments are set forth with particularity in the appended claims. A better understanding of the features and advantages will be obtained by reference to the following detailed description of illustrative embodiments setting forth the principles of the disclosed systems and methods and the accompanying drawings, in which:

[0020] Figure 1A Depicted are exemplary RNA molecules conjugated to chambers as described herein for use in ramp experiments (subjected to multiple cycles of increasing and decreasing force) and constant force experiments (subjected to the same force for a certain amount of time (e.g., 30 seconds to 2 hours) before increasing the force (e.g., a single constant force or a stepped constant force). Single or stepped constant (ramp) forces were applied to the RNA. Figure 1B Shown is an exemplary structure of a nucleic acid comprising a target RNA (XXX) that can be used to analyze the effects of small compound binding according to the embodiments described herein. As shown, a target RNA sequence known to form RNA secondary structure is inserted between two defined sequences on either side of XXX. The two defined sequences are engineered to produce a handle that binds and directs the RNA molecules on the surface of beads and devices. Figure 1CShown are exemplary structures of nucleic acids comprising DNA molecules for analyzing the impact of small compound binding. The known DNA secondary structure-forming DNA (YYY) sequence of interest is inserted between two determined sequences on either side. These sequences are used to produce handles for binding / orienting beads and DNA molecules on the surface. These sequences are used as examples and can be changed.

[0021] FIG. 2A to FIG. 2E The binding affinities of different molecules to the HIV-TAR RNA structure are depicted. Figure 2A The structure of HIV-1 TAR with nucleotides interacting with small molecules as provided in: Chavali SS, Bonn-Breach R, Wedekind JE. (2019) Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery. J. Biol. Chem. 294: 9326-9341 is depicted. Figure 2B ), Netilmicin ( Figure 2C ), kanamycin ( Figure 2D ) and compound 4 (3-amino-6-methyl-N-[3-(trifluoromethyl)phenyl]thieno[2,3-b]pyridine-2-carboxamide) ( Figure 2E ) in the case of, the change of the unfolding force of HIV-TAR molecules. The number (n) of HIV-TAR molecules analyzed under each condition is shown, and the unfolding force of each cycle is standardized relative to the median force of each molecule under control conditions. The force at the maximum distribution of each bead is obtained, and the median force and sem of all beads of each condition are shown. The calculated value KD of each molecule is shown.

[0022] FIG. 3A to FIG. 3K Molecular dynamics of preQ1 riboswitch ligand binding depicted. Figure 3ASchematic representation of the ligand-bound form of the Bsu preQ1 riboswitch and the proposed molecular folding pathway as provided in: Gong Z, Zhao Y, Chen C, Duan Y, Xiao Y (2014) Insights into Ligand Binding to PreQ1Riboswitch Aptamer from Molecular Dynamics Simulations. PLOS ONE 9(3): e92247. Eichhorn, Catherine D. et al. "Structure and function of preQ1riboswitches." Biochimica et biophysica acta 1839 10(2014): 939-950. Figure 3B The transitions of a single preQ1 RNA molecule between unfolded and folded states at 9.6 pN are plotted as the concentration of its ligand preQ1 increases. The traces were fitted using a hidden Markov model. RNA is considered bound to preQ1 when it remains folded for more than 3.3 seconds, and between the two bound states is the time when the RNA is free of preQ1. The association rate (Kon) and dissociation rate (K off ), and the overall KD at 9.6 pN was 73 nM (n=49). Figure 3C The binding kinetics of the preQ1 ligand on the preQ1 RNA secondary structure are shown (k on and k off ), which is an exemplary analysis of Figure 3B Calculations are performed for each trace shown in . Figure 3D Representative binding kinetic curves based on long constant force experiments for measuring Lon of the ligands are shown. Figure 3E The k values ​​based on the ligands used to determine the off Representative binding kinetic curves of long constant force experiments. Figure 3F Depicted are the constant force orders for a single preQ1 RNA molecule as the concentration of preQ1 increases. Each order represents a different force, increasing from left to right. Figure 3G Shown are the transition frequencies between folded and unfolded states plotted against different forces for different preQ1 concentrations. The forces were normalized to the median transition force per bead under control conditions. Figure 3H Graphs showing the unfolding probability of a single molecule at different forces are depicted, and the transition force (50% unfolding probability) can be calculated for different concentrations. Fig. 3I The inflection point half-widths at different concentrations are plotted. KD can be calculated by fitting the inflection point forces at different preQ1 concentrations. Figure 3J PreQ1 and / or Mg are plotted 2+ The relative unfolding force under these conditions. Figure 3K PreQ1 and / or Mg are plotted 2+ The half width of the inflection point in this case.

[0023] FIG. 4A to FIG. 4G The effects of preQ1 RNA mutations on the binding affinity of preQ1 ligands are depicted. Figure 4A The effects of Bsu loop mutations on preQ1 binding are depicted as provided in: Eichhorn, Catherine D. et al. "Structure and function of preQ1 riboswitches." Biochimica et biophysica acta 1839 10 (2014): 939-950. Example WT ( Figure 4B )、C15U( Figure 4C )、U7C+C15U( Figure 4D )、A10G( Figure 4E ) and U6C( Figure 4G ) with increasing concentrations of preQ1 and KD fitting. Figure 4F The effect of different concentrations of preQ1 on mutant A10G is depicted. The unfolding force was normalized to the median force per bead under control conditions. The fraction of cycles where the unfolding force of all beads under control conditions was greater than the 95% confidence interval was calculated for WT, C15U, and U7C+C15U mutants, and this was used to calculate the KD. The threshold force ( FIG. 4B to FIG. 4D For A10G and U6C, the unfolding force of each bead at maximum distribution ( Figure 4E and Figure 4G ). The median of all beads is shown as sem and the number of beads used is also shown. K fitted to each molecule D , and all obtained values ​​are presented in the table (na: not applicable). DETAILED DESCRIPTION

[0024] Overview

[0025] Provided herein are methods for identifying candidates as potential medicines for preventing or treating diseases or conditions directly or indirectly associated with the nucleic acid or protein or its analogs. In some embodiments, these methods can be used to identify candidates for use as medicines against pathogens. In some embodiments, these pathogens include COVID-19 viruses or SARS-COV viruses.

[0026] In some embodiments, the methods described herein include the conformational structure of the target molecule and its ability to interact with the candidate in real time. Therefore, in some embodiments, the methods described herein include considering the primary sequence of the molecule and its structural conformation (folding and / or epigenetic markers) to select binding molecules from the library. This is achieved using the methods described herein, which can modify the secondary structure of the molecule and thus identify the binding of a specific "epitope" or "motif" associated with this secondary structure.

[0027] Since proteins and nucleic acids have secondary and 3D structures, this affects their interactions with other molecules (other nucleic acids or with specific cellular or nuclear components). The presence of epigenetic markers, which can be silent or reinforcing residues, can also affect the ability of candidates to bind to specific "motifs" of target nucleic acids (e.g., DNA or RNA). For example, in some embodiments, the epigenetic marker m6A, which marks RNA, enhances the expression of genes required for embryonic stem cells to correctly differentiate into different cell types. However, in some embodiments, the epigenetic marker m6A restricts the differentiation of blood stem cells. Therefore, in leukemia, a disease in which blood stem cells go wrong, m6A maintains the disease by keeping the cells in a stem cell-like state.

[0028] A variety of conformational structures or folds of double-stranded (ds) and single-stranded (ss) nucleic acids have been identified. Non-limiting examples of conformational structures include hairpins, P-shaped, T-shaped, and candy-shaped. Obtaining these conformational structures is both complex and simple. Therefore, measuring the interaction of molecules with conformational structures remains a challenge. Therefore, the present invention provides devices and methods for detecting and selecting compounds that can affect the function of such target molecules (e.g., nucleic acids).

[0029] Provided herein are methods for detecting structural changes in target molecules (e.g., nucleic acids, proteins, or analogs thereof). Also provided herein are methods for real-time screening and identification in libraries, wherein the library includes molecules that bind to different structural conformations of nucleic acids.

[0030] In some embodiments, these methods include applying a force at one end of a nucleic acid connected to a support (feature of a chamber), and thereby detecting a change in the conformational structure of the nucleic acid. For example, in some embodiments, a change in the force required to unfold (or refold) a nucleic acid in the presence or absence of a binding molecule is detected. Provided herein are methods for screening compounds that bind to a target nucleic acid using the platform described herein. Such methods include fixing the target nucleic acid to a surface of a device at one end and to a magnetic bead at the other end. Figure 1AAs shown, the DNA handle can be used to fix the target RNA to the surface and beads. For example, a feature (e.g., a DNA oligomer) can be chemically connected to the surface, wherein the DNA oligomer has complementarity with the first end of the target nucleic acid. Then, in step (a), a second DNA oligomer conjugated to a biotin moiety and having complementarity with the second end of the target nucleic acid can be added. Alternatively, biotin is present at the 3' end or 5' end of the target RNA itself. Finally, in step (b), magnetic beads with a streptavidin moiety can be added to fix the second end of the target nucleic acid to the magnetic beads. Therefore, in the provided method, step (b) can be performed before step (a), and both are required.

[0031] By providing a fixed target nucleic acid between the device surface and the magnetic beads, a magnetic force can be applied. One way to fix the target nucleic acid is described in the following: Wang, Z., Maluenda, J., Giraut, L. et al. Detection of genetic variation and base modifications at base-pair resolution on both DNA and RNA. Commun Biol 4, 128 (2021). https: / / doi.org / 10.1038 / s42003-021-01648-7, this document is included in this article by reference in its entirety. Applying a magnetic force produces a force along the z axis perpendicular to the device surface. By increasing the magnetic force, the target nucleic acid can be unfolded and then refolded when the magnetic force is released. In a ramp experiment, nucleic acid is subjected to multiple cycles of slowly increasing and reducing force, and the force when the molecule is unfolded or refolded can be determined from one or more sudden changes in the position of the Z beads. In a constant force experiment, nucleic acid is subjected to a constant force for a certain amount of time before rapidly increasing to a higher constant force in a stepwise manner, etc. At one or more of these constant force levels, it is possible to observe transitions of the molecule between folded and unfolded states.

[0032] The methods provided herein include applying a force proportional to the energy required to modify the conformational structure of the nucleic acid in the presence or absence of at least one binding molecule.

[0033] In certain embodiments, the force is between 0.1 piconewtons and 100 piconewtons, between 1 piconewtons and 60 piconewtons, between 1 piconewtons and 35 piconewtons, or between 1 piconewtons and 40 piconewtons or 45 piconewtons. In some embodiments, the force is between 1 piconewtons and 45 piconewtons. In some embodiments, the force is between 1 piconewtons and 40 piconewtons. In some embodiments, the force is between 1 piconewtons and 35 piconewtons.

[0034] In a ramp experiment, a nucleic acid (e.g., RNA) molecule is subjected to multiple cycles of slowly increasing and / or decreasing force, and the value at which the molecule unfolds or refolds can be determined from the abrupt change in the bead position. Upon binding, if the ligand stabilizes or destabilizes the RNA structure, it can cause a change in the unfolding or refolding force.

[0035] In a constant force experiment, the nucleic acid molecule is subjected to the same force for a certain amount of time before the force is increased in a stepwise manner. The equilibrium force is the point at which the molecule spends equal amounts of time in the folded and unfolded states. Ligand binding at this force can cause changes in the folding and unfolding kinetics.

[0036] In some embodiments, the methods described herein are performed at a temperature between 1°C and 45°C, advantageously between 4°C and 37°C, more advantageously between 15°C and 25°C, and even more advantageously at 22°C.

[0037] The binding of molecules (which may include low molecular weight molecules, oligonucleotides or polypeptides, etc.) to target nucleic acids can affect the amount of force required for unfolding or refolding nucleic acid structures. In some cases, the binding of binding molecules can stabilize nucleic acid structures, and thereby increase the force required for unfolding nucleic acid structures. In some cases, the binding of binding molecules can destabilize or change nucleic acid structures, and thereby reduce the force required for unfolding nucleic acid structures. Molecules with a molecular weight of less than 1000 Daltons are considered to be small molecules. Molecules with a molecular weight greater than 1000 Daltons but less than 10000 Daltons can also be tested and considered to be small molecules. The document Synthetic Small-Molecule RNA Ligands: Future Prospects as TherapeuticAgents. MedChemComm. Royal Society of Chemistry, August 14, 2019, pp. 1242-1255. https: / / doi.org / 10.1039 / c9md00195f discloses examples of libraries including a variety of small molecules. Once a binding molecule is selected by the methods described herein or by another method that allows for selection of molecules that bind to nucleic acids, a library comprising analogs and / or chemically modified analogs of the selected member of the initially selected library of binding molecules can be tested by the methods disclosed herein.

[0038] By measuring the changes in unfolding and refolding forces at different concentrations of binding molecules, the binding kinetics of candidate binding molecules can be determined. In addition, multiple nucleic acid molecules can be screened simultaneously at single molecule resolution. This screening provides the ability to detect minimal differences in binding affinity, with improved resolution over other screening techniques. In a specific embodiment, a plurality of molecules in a library means between 1 and 5 x 106 The number of molecules is less than 1x10 8 molecules, for example, in 1x10 8 Up to 1x10 6 molecules, or for example 200,000 molecules, or 4,400 molecules, or even 750 molecules.

[0039] Thus, methods for screening binding and non-binding molecules of nucleic acid molecules are disclosed herein, which methods include: (a) connecting a plurality of nucleic acid molecules to a plurality of beads via a first end of the nucleic acid molecules; (b) connecting a plurality of nucleic acid molecules to a plurality of features of a device via a second end of the nucleic acid molecules; (c) contacting a chamber with a binding molecule library, wherein each member of the binding molecule library is in contact with a single nucleic acid molecule; (d) approaching a magnet to move a plurality of beads along an axis of the chamber relative to the bottom, and measuring changes in the positions of the beads through a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (f) selecting members of the binding molecule library that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (g) modeling the selected members of the binding molecule library to identify chemical tags of the best binders in the binding molecule library.

[0040] system

[0041] Provided herein are systems independently comprising one or more components of a nucleic acid, a DNA handle, a bead, and at least one binding molecule. In some embodiments, the system also includes a binding molecule library. In some embodiments, the system also includes a device (e.g., a chamber) containing a feature, wherein the feature can be used to fix the nucleic acid. In some embodiments, the device can apply a magnetic force to the bead. In some embodiments, the feature is operably connected to a sensor that can measure the impedance caused by the bead moving up and down from the feature.

[0042] Nucleic Acids

[0043] Disclosed herein are nucleic acids (or target nucleic acids) immobilized on features and beads of a device. In some embodiments, the nucleic acid molecule (or target nucleic acid) imparts at least one potential secondary structure. In some embodiments, the secondary structure can be triggered when the molecule binds.

[0044] In some embodiments, the nucleic acid (or target nucleic acid) includes at least one conformational structure. The conformational structure of the nucleic acid (or target nucleic acid) means a secondary or tertiary conformation, such as at least one selected from the conformations of RNA hairpins, P-shaped RNAs, Y-shaped RNAs, candy-shaped RNAs, and combinations thereof. Therefore, in some embodiments, the nucleic acid includes at least one secondary structure. In some embodiments, the nucleic acid includes at least one hairpin structure.

[0045] In particular aspects, the nucleic acid may include an aptamer, in particular a DNA aptamer, an RNA aptamer, an XNA aptamer, or a combination thereof. In particular embodiments, the nucleic acid includes an aptamer specific for the Spike 1 protein of the SARS-Cov-virus, an RNA aptamer specific for the Spike 1 protein of SARS-Cov-2, an XNA aptamer specific for the Spike 1 protein of SARS-Cov-2, or a peptide aptamer specific for the Spike 1 protein of SARS-Cov-2, or a combination thereof.

[0046] In some embodiments, the length of nucleic acid is between 10 bases and 1500 bases. In some embodiments, the length of nucleic acid is between 15 bases and 2000 bases. In some embodiments, the length of nucleic acid molecule is about 20 bases to about 350 bases. In some embodiments, the length of nucleic acid is less than 120 bases. In some embodiments, the length of nucleic acid is greater than 120 bases.

[0047] In some embodiments, the length of nucleic acid is between 10bp and 1500bp. In some embodiments, the length of nucleic acid is between 15bp and 2000bp. In some embodiments, the length of nucleic acid molecule is about 20bp to about 350bp. In some embodiments, the length of nucleic acid is less than 120bp. In some embodiments, the length of nucleic acid is greater than 120bp.

[0048] In some embodiments, the nucleic acid molecules are chemically synthesized. In some embodiments, the nucleic acid molecules are produced by in vitro transcription. In some embodiments, the nucleic acid molecules are produced by polymerase chain reaction. In some embodiments, the nucleic acid molecules are purified from cells.

[0049] In some embodiments, the nucleic acids described herein can be fixed on beads or features of devices by covalent bonding. In some embodiments, the nucleic acids are chemically attached to beads or features of devices. In some embodiments, the nucleic acids are conjugated to molecules comprising a first reactive group. In some embodiments, the first reactive group can react chemically with a second reactive group of beads or features of devices described herein and form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups, which include: (a) DBCO group and azido group; (b) tosyl and amino group; (c) tosyl and sulfhydryl group; (d) epoxy and thiol group; (e) epoxy and amino group; (f) epoxy and hydroxyl group; or (g) chloromethyl and amino group.

[0050] Alternatively, in some embodiments, the nucleic acids described herein can be fixed on a bead or a feature of a device by covalent binding. In some embodiments, the nucleic acid is non-covalently linked (e.g., biotin-streptavidin interaction) to a bead or feature of a device. In some embodiments, the nucleic acid is conjugated to a biotin moiety. In some embodiments, the nucleic acid is conjugated to a biotin moiety at one or both ends of the nucleic acid.

[0051] In some embodiments, nucleic acids as described herein include nucleic acids of interest. In some embodiments, nucleic acids also include directional sequences connected to one or both ends of the nucleic acids of interest. In some embodiments, the directional sequences can hybridize with DNA handles as described herein. Therefore, in some embodiments, the directional sequences include nucleotide sequences complementary to the DNA handles. In some embodiments, the directional sequences include at least 5 bases, at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, at least 50 bases or more. In some embodiments, nucleic acids as described herein can be fixed to the features of beads or devices by hybridizing with DNA handles, wherein the DNA handles are connected to the features of beads or devices. In some embodiments, the directional sequences form the sticky ends of nucleic acids. Therefore, in some embodiments, nucleic acids as described herein including DNA of interest include two chains that are at least partially non-complementary to each other.

[0052] In some embodiments, the nucleic acids of interest described herein are involved in or cause the cause of a disease. In some embodiments, the nucleic acids are involved in preventing or treating a disease. As a control or as a comparison, the same nucleic acid lacking an epigenetic marker or lacking a mutation that causes a disease, or a nucleic acid combined with its complementary RNA or DNA sequence, can be used.

[0053] In some embodiments, the nucleic acid of interest described herein is a viral nucleic acid molecule, and the nucleic acid of interest is isolated from bacteria, preferably from a pathogenic viral agent or from a pathogenic bacterial agent. In some embodiments, the nucleic acid of interest comes from cells isolated from cancer, pathological tissues (such as tumors or amyloid plaques). In some embodiments, the nucleic acid of interest is a viral RNA isolated from an RNA virus, more preferably a viral RNA from a coronavirus, and even more preferably a viral RNA from a SARS or MERS virus or a SARS-COV-2 virus. In some embodiments, the nucleic acid of interest is isolated from an HIV-1 isolate, which is HV-1TAR. In some embodiments, the nucleic acid of interest is a preQ1 riboswitch. In some embodiments, relative to a wild-type preQ1 riboswitch, the preQ1 riboswitch includes a mutation selected from the group consisting of: C15U, U7C, A10G, and U6C. In some embodiments, the nucleic acid of interest is pre-miR-21. In some embodiments, the nucleic acid of interest is DM1.

[0054] In some embodiments, the nucleic acid (or target nucleic acid) includes a nucleic acid of interest, a linker, and a directional sequence. In some embodiments, the nucleic acid molecule includes a construct that includes a nucleic acid of interest, a linker at the 5' end of the nucleic acid and a linker at the 3' end, and two different directional sequences. Examples of linkers and directional sequences are described in Figure 1B and Figure 1C In some embodiments, the nucleic acid of interest comprises DNA, RNA, XNA, aptamer, or a combination thereof or a plurality of nucleic acids that bind to at least one binding molecule of the binding molecule library.

[0055] In some embodiments, the nucleic acid of interest is DNA or RNA, respectively. Figure 1B and Figure 1C As shown, such as (XXX)n and (YYY)n, wherein X (which may be ATG or C) or Y (which may be AUG or C) is a base, n=3 to n=1000, n=5 to n=500, and X and Y may have epigenetic markers (such as methyl groups).

[0056] In some embodiments, the nucleic acid molecule of interest is an aptamer. Thus, in some embodiments, the nucleic acid of interest includes a sequence containing (XXX)n or (YYY)n, wherein X or Y is a base, n=3 to n=1000, n=5 to n=500, Figure 1B and Figure 1C (XXX)n or (YYY)n may include (or consist of) an aptamer.

[0057] Table 1 illustrates exemplary nucleotide sequences of nucleic acids of interest that can be used to analyze the binding of small chemical molecules to secondary structures.

[0058] Table 1. Exemplary nucleic acids of interest

[0059]

[0060] In some embodiments, the nucleic acids described herein include any one of the nucleotide sequences listed in Table 1. In some embodiments, the nucleic acid sequences described herein include at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive nucleotides of any one of the sequences listed in Table 1. In some embodiments, any one of the nucleic acids in Table 1 is conjugated to biotin at the 5' end or the 3' end. Alternatively, in some embodiments, any one of the nucleic acids in Table 1 is conjugated to a molecule comprising a dibenzocyclooctyne (DBCO) group at the 5' end or the 3' end. In some embodiments, any one of the nucleic acids in Table 1 is conjugated to biotin at one end and to a molecule comprising a dibenzocyclooctyne (DBCO) group at the other end.

[0061] In some embodiments, one or two directional sequences are connected to the nucleic acid of interest by a joint. Alternatively, in some embodiments, nucleic acid does not include one or two directional sequences. In such embodiments, the nucleic acid of interest includes a joint at one or both ends of the nucleic acid of interest. Table 2 provides exemplary joint sequences. In some embodiments, the joint includes at least 1 base, at least 2 bases, at least 3 bases, at least 4 bases, at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, at least 9 bases, at least 10 bases, at least 15 bases, at least 20 bases or more. In some embodiments, the joint includes at least 1bp, at least 2bp, at least 3bp, at least 4bp, at least 5bp, at least 6bp, at least 7bp, at least 8bp, at least 9bp, at least 10bp, at least 15bp, at least 20bp or more.

[0062] Table 2. Exemplary linker nucleotide sequences

[0063] Seq ID No. Types of sequences Nucleotide sequence 6 RNA-seq auauau 7 RNA-seq uaacaa 8 RNA-seq auaaua 9 RNA-seq auau 10 RNA-seq uaac 11 DNA sequence ttgtta 12 DNA sequence atatat 13 DNA sequence aaaccc 14 DNA sequence ataata 15 DNA sequence taacaa

[0064] Using linkers on either side of XXX or YYY, a nucleic acid sequence of interest (XXX)n or (YYY)n known to form at least one secondary structure is inserted between two defined sequences (targeting sequences) flanked by the objects [Linker-XXX-Linker] or [Linker-YYY-Linker] (see Figure 1B and Figure 1C ). The identified sequence allows for the targeting of the nucleic acid of interest XXX or YYY and can be single-stranded DNA, single-stranded RNA, etc. For example, the 5' portion of XXX or YYY is linked to a biotin residue. Table 3 provides such Figure 1Band Figure 1C The exemplary nucleic acids described in .

[0065] Table 3. Exemplary nucleotide sequences

[0066]

[0067]

[0068]

[0069] *Bold capital letters represent targeting sequences, lowercase letters represent adapters, and capital letters represent nucleic acids of interest.

[0070] In some embodiments, the nucleic acids described herein include any one of the nucleotide sequences listed in Table 3. In some embodiments, any one of the nucleic acids in Table 3 is conjugated to biotin at the 5' end or the 3' end. In some embodiments, the nucleic acids described herein include any one of the RNA sequences in Table 3 conjugated to biotin at the 5' end. Alternatively, in some embodiments, any one of the nucleic acids in Table 3 is conjugated to a molecule including a dibenzocyclooctyne (DBCO) group, an azido group, a tosyl group, an epoxy group, a chloromethyl group, an amino group, a thiol group, or a hydroxyl group at the 5' end or the 3' end. In some embodiments, any one of the nucleic acids in Table 4 is conjugated to biotin at one end and to a molecule including a dibenzocyclooctyne (DBCO) group at the other end.

[0071] Therefore, the present invention provides the following object: [magnetic bead-linker-streptavidin-biotin-directional sequence linker-(XXX or YYY)n-linker-directional sequence-feature, wherein n is between 5 and 5x10 2 The length of the nucleotide sequence of the present invention is between 15b and 1.5kb, wherein X is a base component of DNA (natural or modified base component of DNA), and Y is a base component of RNA (natural or modified base component of RNA).

[0072] DNA handle

[0073] The DNA handle can be used to fix nucleic acids (e.g., DNA, RNA, or a combination thereof) to the surface of the device and beads. In some embodiments, the nucleic acid is fixed to the surface of the device by the DNA handle, wherein the directional sequence of the nucleic acid hybridizes with at least a portion of the DNA handle. In some embodiments, the DNA handle is 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the directional sequence. In some embodiments, the DNA handle includes at least 5 bases, at least 10 bases, at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, at least 40 bases, at least 45 bases, at least 50 bases or more. In some embodiments, the DNA handle is chemically connected to the surface of the device. In some embodiments, the DNA handle is covalently connected to the surface of the device. In some embodiments, the DNA handle is conjugated to a molecule including a first reactive group, which can react chemically with a second reactive group on the surface of the device to form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups, which include: (a) a DBCO group and an azide group; (b) a tosyl group and an amino group; (c) a tosyl group and a thiol group; (d) an epoxy group and a thiol group; (e) an epoxy group and an amino group; (f) an epoxy group and a hydroxyl group; or (g) a chloromethyl group and an amino group. Thus, in some embodiments, the DNA handle includes a DBCO group and the surface of the device includes an azide group. Conversely, in some embodiments, the DNA handle includes an azide group and the surface of the device includes a DBCO group. In some embodiments, the DNA handle is non-covalently attached to the surface of the device. In such embodiments, the DNA handle is conjugated to a biotin moiety and the surface of the device includes a streptavidin moiety, wherein the streptavidin moiety of the surface of the device is non-covalently bound to the biotin moiety of the DNA handle.

[0074] In some embodiments, the nucleic acid is fixed on the surface of the device, wherein the terminal nucleotide sequence of the nucleic acid is chemically connected to the surface of the device. In some embodiments, the terminal nucleotide sequence of the nucleic acid is covalently connected to the surface of the device. In some embodiments, the nucleic acid is conjugated to a molecule including a first reactive group, which can react chemically with a second reactive group on the surface of the device to form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups, which include: (a) DBCO group and azido group; (b) tosyl group and amino group; (c) tosyl group and sulfhydryl group; (d) epoxy group and thiol group; (e) epoxy group and amino group; (f) epoxy group and hydroxyl group; or (g) chloromethyl and amino group. Alternatively, in some embodiments, the nucleic acid is fixed on the surface of the device, wherein: the nucleic acid is conjugated to a biotin moiety; the surface of the device includes a streptavidin moiety; and the streptavidin moiety on the surface of the device is non-covalently bound to the biotin moiety of the nucleic acid.

[0075] In some embodiments, the nucleic acid is immobilized on a bead, wherein the bead includes a streptavidin portion, and the nucleic acid is conjugated to a biotin portion that is non-covalently bound to the streptavidin portion of the bead. Alternatively, in some embodiments, the nucleic acid is chemically attached to the bead. Thus, in some embodiments, the nucleic acid is covalently attached to the bead. In some embodiments, the nucleic acid is conjugated to a molecule including a first reactive group that is capable of chemically reacting with a second reactive group of the bead to form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups that include: (a) a DBCO group and an azido group; (b) a tosyl group and an amino group; (c) a tosyl group and a thiol group; (d) an epoxy group and a thiol group; (e) an epoxy group and an amino group; (f) an epoxy group and a hydroxyl group; or (g) a chloromethyl group and an amino group.

[0076] In some embodiments, the nucleic acid is immobilized on the bead by a DNA handle, wherein the terminal nucleotide sequence of the nucleic acid hybridizes to at least a portion of the DNA handle. In some embodiments, the DNA handle is 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the terminal nucleotide sequence. In some embodiments, the DNA handle is covalently attached to the bead. In some embodiments, the DNA handle is conjugated to a molecule comprising a first reactive group that is capable of chemically reacting with a second reactive group of the bead to form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups, which include: (a) a DBCO group and an azide group; (b) a tosyl group and an amino group; (c) a tosyl group and a thiol group; (d) an epoxy group and a thiol group; (e) an epoxy group and an amino group; (f) an epoxy group and a hydroxyl group; or (g) a chloromethyl group and an amino group. Alternatively, in some embodiments, the DNA handle is non-covalently attached to the bead. In such embodiments, the DNA handle is conjugated to a biotin moiety and the bead comprises a streptavidin moiety, wherein the streptavidin moiety of the bead is non-covalently bound to the biotin moiety of the DNA handle.

[0077] Table 4 provides exemplary nucleotide sequences of DNA handles that can be used to immobilize nucleic acids (eg, DNA, RNA, or a combination thereof).

[0078] Table 4. Exemplary DNA handles

[0079]

[0080] In some embodiments, the DNA handle described herein comprises any one of the nucleotide sequences listed in Table 4. In some embodiments, the DNA handle described herein comprises at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 consecutive nucleotides of any one of the sequences listed in Table 4. In some embodiments, any one of the DNA handles of Table 4 is conjugated to biotin at the 5' end or the 3' end. Alternatively, in some embodiments, any one of the DNA handles of Table 4 is conjugated to a molecule comprising a dibenzocyclooctyne (DBCO) group at the 5' end or the 3' end. In some embodiments, one or more bases of the DNA handle sequence are chemically modified. In some embodiments, the modification of the DNA handle sequence comprises adding an active primary amino group to one or more nucleotides. In some embodiments, the modification can be performed by an amino dT C6 reagent. Thus, in some embodiments, the modification is a BCN-I (bicyclooctyne) modification. In some embodiments, the DNA handle comprising the modified nucleotides comprises the nucleotide sequence of SEQ ID NO:47.

[0081] Beads

[0082] In some embodiments, the beads described herein are magnetic beads. In some embodiments, the beads are attached to a DNA handle and / or nucleic acid. In some embodiments, the magnetic beads include a non-conductive polymer, silicon, glass, or resin.

[0083] In some embodiments, the beads include a streptavidin moiety that is capable of binding to a biotin moiety of a DNA handle and / or nucleic acid. Alternatively, in some embodiments, the beads include a first reactive group that is capable of chemically reacting with a second reactive group of a DNA handle or nucleic acid to form a covalent bond. In some embodiments, the chemical reaction occurs between two reactive groups, which include: (a) a DBCO group and an azide group; (b) a tosyl group and an amino group; (c) a tosyl group and a thiol group; (d) an epoxy group and a thiol group; (e) an epoxy group and an amino group; (f) an epoxy group and a hydroxyl group; or (g) a chloromethyl group and an amino group. Thus, in some embodiments, the beads include a dibenzocyclooctyne (DBCO) group, an azide group, a tosyl group, an epoxy group, a chloromethyl group, an amino group, a thiol group, or a hydroxyl group.

[0084] In some embodiments, the diameter of the magnetic beads is in the following range: 0.1μm to 10μm, 0.3μm to 10μm, 0.5μm to 10μm, 1μm to 10μm, 2μm to 10μm, 5μm to 10μm, 0.1μm to 5μm, 0.3μm to 5μm, 0.5μm to 5μm, 1μm to 5μm, 2μm to 5μm, 0.1μm to 2μm, 0.3μm to 2μm, 0.5μm to 2μm, 1μm to 2μm, 0.1μm to 1μm, 0.3μm to 1μm, 0.5μm to 1μm, 0.1μm to 0.5μm, 0.3μm to 0.5μm or 0.1μm to 0.3μm. In some embodiments, the diameter of the magnetic beads is 0.3 μm, 0.5 μm, 1.04 μm, 2.8 μm, or 5.5 μm.

[0085] Device

[0086] In some embodiments, the device described herein includes an actuator, a sensor, and a chamber. In some embodiments, the actuator is suitable for moving the bead in a direction of motion relative to the surface of the device. In some embodiments, the sensor is suitable for measuring the distance between the bead and the surface. In some embodiments, the chamber includes an axis extending along the direction of motion of the bead and a bottom formed by a feature (e.g., a surface).

[0087] In some embodiments, bead as described herein is magnetic bead, and magnet is used to produce the force that changes the position of magnetic bead in chamber.In some embodiments, the force of magnet relative to bead is constant.In some embodiments, increase the force (slope) of magnet relative to bead.Whether protein, antibody, aptamer are connected to nucleic acid molecule, it is suitable to allow to apply force to unfold nucleic acid other devices.Use the sensor of device to measure the change of the position of bead in chamber.

[0088] In some embodiments, the features as described herein may represent at least 10,000 features. In some embodiments, the sensor includes a camera, advantageously a CMOS camera, which is capable of collecting photons reflected from the beads. In some embodiments, the sensor includes a CMOS capable of measuring the impedance caused by the up and down movement of the beads from the features. In some embodiments, the nucleic acid molecules are DNA molecules. In some embodiments, the nucleic acid molecules are RNA molecules. In some embodiments, the nucleic acid molecules include DNA / RNA hybrid molecules. In some embodiments, a plurality of nucleic acid molecules include the same nucleic acid molecules. In some embodiments, a plurality of nucleic acid molecules are different nucleic acid molecules. In some embodiments, a plurality of nucleic acid molecules are different from each other by at least one or two bases.

[0089] Conductive solution

[0090] In some embodiments, the systems described herein include a conductive solution disposed in a chamber described herein. In some embodiments, the chamber includes a sensor adapted to measure the impedance of the chamber. In some embodiments, the impedance is a function of the distance between the beads and a feature (e.g., a surface). In some embodiments, the conductive solution may have a -7 S / cm to 10 1 S / cm or 10 -3 S / cm to 10 -2 The conductivity is between S / cm.

[0091] Binding molecules

[0092] In some embodiments, the system described herein includes a binding molecule or a binding molecule library. In some embodiments, the binding molecule library includes analogs and / or chemically modified analogs of selected members of the binding molecule library. In some embodiments, the binding molecule can be combined with nucleic acids as described herein. In some embodiments, the binding molecule can produce an unfolding force of nucleic acid, which is greater than or less than the unfolding force of the nucleic acid when there is no binding molecule. In some embodiments, the unfolding force depends on the amount of the binding molecule.

[0093] In some embodiments, the binding molecule library comprises aptamers, in particular DNA aptamers, RNA aptamers, XNA aptamers, peptide aptamers or combinations thereof. Aptamers, first described as artificial oligonucleotides that bind to one or more specific target molecules, exhibit a range of affinities (K D Aptamers can be thought of as chemical antibodies.

[0094] A skilled person can easily find other examples of binding molecule libraries. The following binding molecule libraries are examples of binding molecule libraries suitable for the claimed methods: https: / / enamine.net / compound-libraries / targeted-libraries / ma, https: / / lifechemicals.com / screening-libraries / targeted-and-focused-screening-libraries / ma-focused-library, https: / / www.discoverx.com / products-applications / prod-ligands-inhibitors, and https: / / spirochem.com / catalog.

[0095] In some embodiments, binding molecules as described herein can stabilize the structure of nucleic acid, and thus increase the power required for the structure of unfolded nucleic acid. In some embodiments, binding molecules as described herein can destabilize or change the structure of nucleic acid, and thus reduce the power required for the structure of unfolded nucleic acid. Molecules with a molecular weight less than 1000 daltons are considered to be small molecules. Molecules with a molecular weight greater than 1000 daltons but less than 10000 daltons can also be tested and are considered to be small molecules.

[0096] Binding molecules may include molecules that bind to a target nucleic acid (or nucleic acids) to a sufficient degree to affect the folding or unfolding of the target nucleic acid. Such binding molecules may have a K of D: about 0.01nM, 0.02nM, 0.03nM, 0.04nM, 0.05nM, 0.06nM, 0.07nM, 0.08nM, 0.09nM, 0.1nM, 0.2nM, 0.3nM, 0.4nM, 0.5nM, 0.6nM, 0.7nM, 0.8nM, 0.9nM, 1nM, 2nM, 3nM, 4nM, 5nM, 6nM, 7nM, 8nM, 9nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 70 0nM, 800nM, 900nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20 μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 200μM, 300μM, 400μ M, 500μM, 600μM, 700μM, 800μM, 900μM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM , 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM.

[0097] Binding molecules as disclosed herein may include small molecules, chemicals, polypeptides, or other polynucleotides as demonstrated in the examples. In a specific embodiment of the invention, binding molecules as disclosed herein may include polypeptides or other polynucleotides as demonstrated in the examples. In some embodiments, the binding molecule is a medicine.

[0098] Methods for identifying chemical markers

[0099] Disclosed herein is a method for chemically tagging the best binder in a binding molecule library of nucleic acid molecules, the method comprising: (a) attaching a first end of the nucleic acid molecule to a bead; (b) attaching a second end of the nucleic acid molecule to a feature of a device in a chamber, the chamber comprising a bottom surface and an axis, the device also comprising a sensor for detecting the position of the bead; (c) optionally applying a force to the nucleic acid to unfold the nucleic acid and refold the nucleic acid as a control; (d) contacting the nucleic acid molecule with a molecular library, wherein each member of the binding molecule library is in contact with a single nucleic acid molecule; (e) applying a force to the nucleic acid to unfold the nucleic acid; (f) selecting members of the binding molecule library that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; (g) modeling the selected members of the binding molecule library to identify the chemical tag of the best binder in the binding molecule library; and (g) optionally, testing the activity of the best binder or (g) and their analogs / chemically modified binders as medicines.

[0100] The present invention also discloses a method for chemically labeling the best binder in a binding molecule library of nucleic acid molecules, the method comprising: (a) connecting multiple nucleic acid molecules to multiple beads through the first end of the nucleic acid molecule; (b) connecting multiple nucleic acid molecules to multiple features of a device through the second end of the nucleic acid molecule; (c) contacting a chamber with the binding molecule library, wherein each member of the binding molecule library is in contact with a single nucleic acid molecule; (d) approaching a magnet to move multiple beads along the axis of the chamber relative to the bottom surface of the feature, and measuring the change in the position of the beads through a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (f) selecting members of the binding molecule library that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (f) modeling the selected members of the binding molecule library to identify the chemical labeling of the best binder in the binding molecule library.

[0101] Methods and systems for determining binding kinetics

[0102] Disclosed herein are methods for determining the binding kinetics of binding molecules to nucleic acid molecules, which methods include: (a) connecting one end of the nucleic acid molecule to a bead; (b) connecting one end of the nucleic acid molecule to a feature of a device in a chamber, the chamber comprising a bottom surface and an axis, the device also comprising a sensor for detecting the position of the bead; (c) contacting the nucleic acid molecule with increasing concentrations of the binding molecule; (d) applying a force to the nucleic acid to unfold the nucleic acid; (e) determining the unfolding force of the nucleic acid molecule that varies according to the concentration of the binding molecule; and (f) calculating the binding kinetics of the binding molecule to the nucleic acid molecule based on the change in the unfolding force that varies according to the amount of the binding molecule.

[0103] The present invention also discloses methods for determining the binding kinetics of binding molecules to nucleic acid molecules, which methods include: (a) connecting multiple nucleic acid molecules to multiple beads via the first ends of the nucleic acid molecules; (b) connecting multiple nucleic acid molecules to multiple features of a device via the second ends of the nucleic acid molecules; (c) contacting a chamber with an increasing concentration of binding molecules; (d) applying a magnetic field sufficient to move each of the multiple beads relative to the bottom surface of the feature of the chamber, and measuring the change in the position of the beads based on the movement of the beads relative to the bottom surface through a sensor of the device; (e) determining the unfolding force of the nucleic acid molecules that varies according to the concentration of the binding molecules; and (f) calculating the binding kinetics of the binding molecules to the nucleic acid molecules based on the change in the unfolding force that varies according to the amount of the binding molecules.

[0104] Also disclosed herein is a system for determining the binding kinetics of a binding molecule to a nucleic acid molecule. In some embodiments, the system includes: (a) a plurality of nucleic acid molecules connected to a plurality of beads by one end of the nucleic acid molecule; (b) a plurality of nucleic acid molecules connected to a plurality of features of a device by a second end of the nucleic acid molecule; (c) a chamber having a binding molecule library, wherein each member of the binding molecule library of increasing doses is contacted with a plurality of single nucleic acid molecules in a single chamber; (d) a magnet that moves a plurality of beads along the axis of the chamber relative to the bottom surface; and (e) a sensor that measures changes in the bead position of the device based on the movement of the beads relative to the bottom surface.

[0105] Screening methods

[0106] In some embodiments, a screening method is provided, the screening method comprising: (a) connecting a nucleic acid molecule having at least two ends to a magnetic bead at one end; (b) connecting the other end of the nucleic acid molecule to a feature of a device comprising a sensor for detecting a position change of the magnetic bead; (c) applying a force by bringing a magnet close to the magnetic bead to change the position of the magnetic bead relative to the bottom surface and unfolding the nucleic acid; (d) optionally, before or after refolding the nucleic acid, contacting the nucleic acid with at least one molecule; (e) quantifying the change in the magnetic bead position or the unfolding force before and after contacting the nucleic acid molecule with a binding molecule library; and (f) selecting a binding molecule that produces an unfolding force of a nucleic acid molecule, the unfolding force being greater than or less than the unfolding force of the nucleic acid molecule in the absence of a binding molecule. In some embodiments, the method further comprises repeating steps (a) to (d) with a binding molecule library, the binding molecule library comprising analogs and / or chemically modified analogs of selected members of the binding molecule library. In some embodiments, the method further comprises testing the activity of the selected binding molecule and / or their analogs / modified forms as medicines.

[0107] Disclosed herein are methods for screening or identifying binding molecules of nucleic acid molecules, which methods include: (a) orienting a nucleic acid molecule or multiple nucleic acid molecules having a 5' end and a 3' end in a device by attaching the 5' end to a bead and the 3' end to the bottom surface of the device, the device also including a sensor for detecting the position of the bead; (b) contacting the nucleic acid molecule with a molecular library before, during or after applying a force to the nucleic acid by bringing a magnet close to the magnetic bead; and (c) measuring the change in the position of the magnetic bead based on the movement of the bead relative to the bottom surface by the sensor of the device.

[0108] In some embodiments, the method comprises: (a) measuring conformational changes of the nucleic acid caused by applying forces to the nucleic acid molecule before, during, and after the nucleic acid is contacted with a plurality of molecules and / or increasing doses of the molecule; and (b) selecting a molecule that binds directly or indirectly to the nucleic acid. In some embodiments, the method further comprises testing the molecule as a medicine for preventing and / or treating a disease associated with the nucleic acid.

[0109] In some embodiments, by using a magnet to apply force (advantageously a series of constant forces or a series of increasing forces) to the nucleic acid molecules and measuring the movement of the beads connected to the nucleic acid, the conformational changes of the nucleic acid connected to the magnetic beads are performed in real time. This step is repeated before, after or when the nucleic acid contacts multiple molecules and / or increased doses of molecules. Therefore, in some embodiments, the nucleic acid described herein includes at least one conformational structure alone or after contact with a molecule.

[0110] In some embodiments, the method includes: (a) connecting the 5' end of the nucleic acid molecule to a bead; (b) connecting the 3' end of the nucleic acid molecule to a feature of a device in a chamber, the chamber including a bottom surface and an axis, the device also including a component for detecting the binding of a molecule according to a conformational change of the nucleic acid; (c) before or during the application of force to the nucleic acid, contacting a plurality of molecules from a library with the nucleic acid; (d) identifying a molecule that produces an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (e) testing analogs, chemically modified molecules. In some embodiments, the method also includes testing the activity of the selected binding molecule as a medicine. In some embodiments, by measuring the Ka, Kon and / or K of the selected binding molecule O ff to determine binding.

[0111] Disclosed herein are methods for identifying binding molecules of nucleic acid molecules including a first end and a second end, the methods comprising: (a) connecting a plurality of nucleic acid molecules to a plurality of beads by the first end of the nucleic acid molecule; (b) connecting a plurality of nucleic acid molecules to a plurality of features of a device by the second end of the nucleic acid molecule; (c) contacting a chamber with a binding molecule library, wherein each member of the binding molecule library of increasing doses is contacted with a single nucleic acid molecule; (d) approaching a magnet to move a plurality of beads relative to the bottom along the axis of the chamber, and measuring changes in the positions of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (f) distinguishing members of the binding molecule library that produce an unfolding force of the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (g) modeling selected members of the binding molecule library to identify chemical markers of the best binders in the binding molecule library. In some embodiments, the method also includes testing the activity of the identified molecule in preventing or treating a disease directly or indirectly associated with a bound or unbound RNA.

[0112] In some embodiments, the method includes: (a) connecting a nucleic acid molecule including two ends to a bead by one end; (b) connecting the second end of the nucleic acid molecule to a feature of a device; (c) contacting a chamber with a binding molecule library, wherein each member of the binding molecule library is in contact with a plurality of single nucleic acid molecules in a single chamber; (d) approaching a magnet to move the bead along the axis of the chamber relative to the bottom surface, and measuring changes in the position of the bead by a sensor of the device based on the movement of the bead relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; and (f) selecting a member of the binding molecule library that produces an unfolding force of the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule. In some embodiments, the method also includes testing the activity of the identified binding molecule as a medicine for preventing or treating a disease directly or indirectly associated with the nucleic acid.

[0113] In some embodiments, diseases directly associated with nucleic acids include diseases associated with exposure of the body or cells to nucleic acids or to infectious pathogens comprising the nucleic acids.

[0114] In some embodiments, diseases indirectly associated with nucleic acids include diseases associated with exposure of the body or cells to nucleic acids and / or to products derived from nucleic acids by transcription, translation, replication, or a combination thereof.

[0115] The methods provided herein can detect and / or identify molecules that bind to a conformational structure of a nucleic acid or to a conformational structure close to the nucleic acid. The nucleic acid can be a plurality of identical nucleic acids or a plurality of different nucleic acid molecules. A plurality of nucleic acids are bound to a plurality of beads. In a particular embodiment, the nucleic acid molecule includes several motifs that can be recognized by an aptamer. In a particular embodiment, the nucleic acid molecule can be an aptamer.

[0116] In some embodiments, the method further comprises the step of contacting a nucleic acid comprising (or consisting of) an aptamer and analyzing the binding of a small molecule, a protein (such as an enzyme) that interacts with the nucleic acid. In some embodiments, the method further comprises the step of contacting the nucleic acid with a molecule x (in the presence or absence of a molecule from a library, before, during or after unfolding the nucleic acid), the molecule x may be a protein that blocks non-specific binding (may be bovine serum albumin (BSA)), an antibody, in particular an antibody specific for an epigenetic marker, an antibody specific for a methyl or acetyl group, an enzyme that interacts with the nucleic acid at one point in translation, a cofactor. In some embodiments, the method further comprises the step of contacting the nucleic acid with a molecule x (in the presence or absence of a molecule from a library, before, during or after unfolding the nucleic acid), the molecule x may be an aptamer. This optional step of contacting the nucleic acid with a molecule x may be performed before, during or after contacting with a molecule from the library.

[0117] In the case of multiple identical nucleic acids, a chemical labeling of the best binder in the binding molecule library can be provided. Therefore, the present invention also discloses a method for identifying the chemical labeling of the best binder in the binding molecule library of nucleic acid molecules, the method comprising: (a) contacting a single nucleic acid molecule with the binding molecule library, wherein each member of the binding molecule library is in contact with a single nucleic acid molecule; (b) approaching a magnet to move a plurality of beads relative to the bottom surface along the axis of the chamber, and measuring the change in the position of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface; (c) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (d) selecting a member of the binding molecule library that generates an unfolding force of the nucleic acid molecule, the unfolding force being greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; and (e) modeling the selected members of the binding molecule library to identify the chemical labeling of the best binder in the binding molecule library.

[0118] As disclosed herein, multiple binding molecules can be screened for the ability to bind to a target nucleic acid molecule. In some cases, a plurality of identical target nucleic acid molecules can be immobilized using the platform device described herein for screening. In some cases, a plurality of different target nucleic acid molecules can be immobilized using the platform device described herein for screening. The unfolding and refolding forces can be calculated using a ramp force experiment as described in the following examples. The binding kinetics (e.g., K on and K off ) can be calculated using a constant force experiment as described in the Examples below. The effect of ligand binding on the unfolding and refolding forces can be used to distinguish molecules that bind to a target nucleic acid with different binding affinities.

[0119] In some cases, multiple candidates can be screened for the ability to bind to the target nucleic acid. In some cases, the test can be performed on the same target molecule (when this is a single molecule platform), and this process can be non-destructive. For example, a chamber containing a single target nucleic acid can be screened with a separate candidate, and the ability to bind to the target nucleic acid can be determined by the change of unfolding and refolding power when contacting the candidate. The candidate that can be combined with the target nucleic acid (that is, those candidates that have the greatest impact on unfolding or refolding power, as measured by impedance) or measured using an optical device) can be quickly identified. In addition, the method described herein can distinguish the binding agent with a dynamic range of binding affinity. For example, the methods described herein can identify binders with a KD of about 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 50 0nM, 600nM, 700nM, 800nM, 900nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μ M, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM, 200μM, 300μ M, 400μM, 500μM, 600μM, 700μM, 800μM, 900μM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM. In addition, the methods described herein can distinguish between binders with less than 30-fold, less than 20-fold, less than 10-fold, less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1-fold, less than 0.9-fold, less than 0.8-fold, less than 0.7-fold, less than 0.6-fold, less than 0.5-fold, less than 0.4-fold, less than 0.3-fold, less than 0.2-fold, or less than 0.1-fold differences in unfolding or refolding forces. In some embodiments, the methods described herein can distinguish between binders with about 0.1-fold to about 30-fold differences in unfolding or refolding forces.

[0120] As described herein, a target nucleic acid may include a target DNA, target RNA, or hybrid involved in a disease or condition. For example, a target nucleic acid may include HIV-1 TAR, preQ1 riboswitch, pre-miR-21, MYC, APOC3, DM1, C9orf72, MAPT, or FMRI. Alternatively and in addition, a target nucleic acid may include HIV-1 TAR, preQ1 riboswitch, pre-miR-21, MYC, APOC3, DM1, C9orf72, MAPT, or FMRI, an aptamer that binds to the SARS-Cov-2 spike protein.

[0121] The example of such nucleic acid is in Table 3. Target nucleic acid can be chemically synthesized before fixing, can be recombinantly produced in microorganisms, or can be produced by in vitro transcription. In some cases, nucleic acid molecules can be produced by polymerase chain reaction. The size of such target nucleic acid can be in the following range: about 10bp to about 1000bp or about 10bp to 1500bp (for example, about 20bp to about 350bp, about 50bp to about 200bp, about 75bp to about 150bp, etc.). In some embodiments, the size of such target nucleic acid can be in the following range: about 10 bases to about 1000 bases or about 10 bases to 1500 bases (for example, about 20 bases to about 350 bases, about 50 bases to about 200 bases, about 75 bases to about 150 bases, etc.).

[0122] In some embodiments, the target nucleic acid can be a nucleic acid with natural modification. In fact, the nucleic acid with natural modification (post-transcription / epigenetic) can interact with the binding molecule differently from the nucleic acid without modification. Therefore, the method described herein can be used to distinguish modified and unmodified nucleic acids. This method can be used to screen the modification involved in a disease or condition. Specifically, nucleic acid can be separated from a subject suspected of having a disease or condition, which can be fixed in a feature as described herein. When the unfolding and refolding power of the modified and unmodified target in the presence of a given ligand is known, the unfolding and refolding power of the nucleic acid separated from the subject in the presence of the ligand and the presence or absence of the modification can be quickly determined by measuring the unfolding and refolding power of the nucleic acid separated from the subject in the presence of the ligand and comparing with the standard.

[0123] Reagent test kit

[0124] Also disclosed herein is a system for practicing the methods disclosed herein. In some cases, a kit may include a system disclosed herein. In some cases, an apparatus may include a system disclosed herein. For example, a system may include (a) multiple nucleic acid molecules connected to multiple magnetic beads by one end of a nucleic acid molecule; (b) multiple nucleic acid molecules connected to multiple features of a device by a second end of a nucleic acid molecule; (c) a chamber with a binding molecule library, wherein each member of the binding molecule library is in contact with multiple single nucleic acid molecules in a single chamber; and (d) a magnet that moves multiple beads along the axis of the chamber relative to the bottom surface of the feature; and (e) a sensor that measures the change in the bead position of the device based on the movement of the beads relative to the bottom surface. In some cases, the system can be used to screen binding molecules for nucleic acid molecules. In some cases, the system can be used to determine the binding kinetics of binding molecules to nucleic acid molecules. In some cases, a kit may include a container and instructions for use. In addition, kits are provided herein, comprising: (a) one or more identical or different nucleic acid molecules comprising a directional sequence at one end and a biotin moiety at the other end; (b) a DNA handle comprising a first reactive group, wherein the DNA handle is capable of hybridizing with the directional sequence; (c) a feature of a device comprising a second reactive group capable of chemically reacting with the first reactive group; and (d) a bead or multiple beads comprising a streptavidin moiety capable of binding to a biotin moiety. Kits are also provided herein, comprising: (a) one or more identical or different nucleic acid molecules comprising a first directional sequence at one end and a second directional sequence at the other end; (b) a first DNA handle comprising a biotin moiety, wherein the first DNA handle is capable of hybridizing with the first directional sequence; (b) a second DNA handle comprising a first reactive group, wherein the DNA handle is capable of hybridizing with the second directional sequence; (c) a feature of a device comprising a second reactive group capable of chemically reacting with the first reactive group; and (d) a bead or multiple beads comprising a streptavidin moiety capable of binding to a biotin moiety. In some embodiments, the kit further comprises a library of binding molecules. In some embodiments, the binding molecule library includes a small molecule library, a protein library, an antibody library, an aptamer library, and advantageously includes a library of binding molecules. In some embodiments, the kit also includes a buffer containing a divalent cation in the range of 1 μM to 1 mM. In some embodiments, the divalent cation includes Mg. 2+ , Mn 2+ 、Zn 2+ , Fe 2+ , Cu 2+ or Cd 2+ In some embodiments, the chemical reaction is an esterification reaction. In some embodiments, the chemical reaction is click chemistry.

[0125] As another aspect, a kit (or system) is provided herein, comprising: (a) one or more identical or different nucleic acid molecules, covalently attached to a bead at one end using a linker, DNA handle and / or click, advantageously using a biotin and streptavidin moiety; (b) the nucleic acid is attached at the other end to a feature at the bottom surface of a chamber in a device; (c) the feature may be attached at the other end to the bottom surface of the chamber using a linker, DNA handle and / or click; (d) the device further comprising a sensor for detecting the position of the bead; a magnet; (e) a binding molecule library, such as a small molecule library, a protein library, an antibody library, an aptamer library; (f) a buffer, advantageously comprising a divalent cation, such as Mg 2+ , Mn 2+ 、Zn 2+ , Fe 2+ , Cu 2+ 、Cd 2+ , preferably Mg 2+ In some embodiments, the buffer contains less than 10 mM Mg 2+ , less than 5mM Mg 2 + , advantageously >0.01 mM Mg 2+ and less than 10 mM Mg 2+ , more advantageously 1 mM Mg 2+ .

[0126] In some embodiments, a kit is provided herein, comprising: (a) a nucleic acid molecule or a plurality of nucleic acids; (b) a bead or a plurality of beads; (c) at least two adapters, DNA handles and / or clicks; and (d) two directional sequences. In some embodiments, the beads are magnetic beads.

[0127] In some embodiments, provided herein is a device comprising: a feature at the bottom surface of a chamber, the device further comprising a sensor, advantageously a magnetic sensor; at least one library of binding molecules; and a buffer.

[0128] platform

[0129] Screening methods as described herein can utilize devices for measuring the unfolding and refolding of target nucleic acids. The device may include a device described in U.S. Patent No. 10,933,416, which is incorporated by reference in its entirety. For example, one of the devices and / or a part of the device that can be used to achieve this unfolding of nucleic acids and detect it is disclosed in patent WO2016177869. Therefore, the optical device that can detect the unfolding of the sampled nucleic acid can be those described in the following: EP3181703B or EP3990958, technical EP3505641, preferably in EP3914949, EP3803492; All of these documents are fully included by reference.

[0130] Other embodiments of the device as described herein may also include: (a) a magnetic bead (e.g., a magnetic bead "MyOne" produced by Invitrogen, having a diameter of 1.04 μm; a magnetic bead "M270" produced by Invitrogen, having a diameter of 2.8 μm; a magnetic bead "M450" produced by Invitrogen, having a diameter of 5.5 μm; a magnetic bead "Ademtech 500" produced by Ademtech, having a diameter of 0.5 μm; a magnetic bead "Ademtech 300" produced by Ademtech, having a diameter of 0.3 μm) to which one end of the target nucleic acid can be attached; (b) a surface (e.g., silicon, glass, a non-conductive polymer or resin) to which a second end of the target nucleic acid can be attached; (c) an actuator adapted to move the bead in a direction of motion relative to the surface of the device; (d) a sensor adapted to measure the distance between the bead and the surface; (e) a chamber having an axis extending along the direction of motion of the bead and a bottom formed by the surface; and (f) a conductive solution disposed in the chamber, wherein the conductivity of the conductive solution can be within 10 -7 S / cm to 10 1 S / cm or between 10 -3 S / cm to 10 -2 S / cm, wherein the sensor is adapted to measure the impedance of the chamber, wherein the impedance is a function of the distance between the bead and the surface.

[0131] Also disclosed herein is a system or device (platform) for real-time selection of molecules bound to nucleic acid conformational states, including a plurality of magnetic beads, a magnet, features in a chamber, and a sensor for detecting movement of beads attached to nucleic acids, the movement varying according to conformational changes of the nucleic acids. In some embodiments, the chamber comprises a bottom surface and an axis.

[0132] In some embodiments, the apparatus may include the apparatus described in U.S. Pat. No. 9,933,609, which is incorporated by reference in its entirety. In some embodiments, the apparatus may include the apparatus described in U.S. Pat. No. 10,209,505, which is incorporated by reference in its entirety. The apparatus as described herein may include: a) an objective lens for collecting optical radiation diffused by an object, the imaging system having an optical axis extending parallel to a first axis; b) a transmission mask having at least a first aperture and a second aperture, the first aperture and the second aperture being spaced apart from each other along a second axis perpendicular to the first axis, the transmission mask being arranged so that a first portion of radiation and a second portion of radiation diffused by the object pass through the first aperture and the second aperture, respectively, while blocking a portion of radiation emitted by the light source that is not diffused by the object; and c) a detector adapted to generate an image comprising a first light spot and a second light spot, the first light spot and the second light spot representing a first portion and a second portion of radiation impinging on a plane of the detector, wherein a change in the position of the object relative to the object plane of the imaging system along the first axis causes a change in the position of the first light spot and the second light spot relative to each other along the second axis.

[0133] In some embodiments, the sensor may include: a main electrode, which is located at the top of the chamber and in contact with the conductive solution, to which a known potential is applied; a second electrode, which is located at the bottom of the chamber and carries a surface to which molecules can be attached; and an electronic circuit suitable for measuring the current flowing between the electrodes, the electronic circuit comprising: a current-voltage amplifier connected to the second electrode; a voltmeter suitable for measuring the output voltage of the current-voltage amplifier; and a computing circuit suitable for calculating the impedance of the well from the measured voltage. In some cases, the sensor may include a camera CMOS capable of collecting photons reflected from the beads. In some cases, the sensor may include a CMOS capable of measuring the impedance caused by the up and down movement of the beads from the features. In some cases, the chamber is set in a device configured to measure the displacement of the beads of nucleic acid according to the concentration fluctuation of the binding molecule.

[0134] The actuator may include at least one permanent magnet, which may be controlled to move along the XX axis translation. The actuator may include two permanent magnets, which are located at equal distances from the XX axis and align their magnetic poles perpendicular to the XX axis, with the north pole of one magnet facing the south pole of another magnet. In some cases, the force of the magnet relative to the bead is constant. In some cases, the force of the magnet relative to the bead is increased or decreased. In some embodiments, the force of the magnet is at least 1 piconewton, at least 5 piconewtons, at least 10 piconewtons, at least 20 piconewtons, at least 35 piconewtons, at least 70 piconewtons or at least 95 piconewtons. In some embodiments, the force of the magnet is no more than 100 piconewtons. The force of the magnet in the presence of a binding molecule is compared with the force when there is no binding molecule. An example of a magnet relative to the force of a bead can be 1 piconewton to 35 piconewtons or more and at most 100 piconewtons.

[0135] In some embodiments, attachment of beads can include biotin-streptavidin binding. In some cases, biotin is conjugated to one end of the nucleic acid and streptavidin is conjugated to the beads.

[0136] In some embodiments, nucleic acid is connected to a plurality of features and comprises that DNA oligonucleotide is covalently connected to the bottom of each feature. In some cases, DNA oligonucleotide has complementarity with the second end of nucleic acid molecule. In some embodiments, a plurality of nucleic acid molecules are anchored to the bottom of a plurality of features. In some cases, each feature of chamber comprises a single nucleic acid molecule sequence in a plurality of nucleic acid molecule sequences.

[0137] In some embodiments, a single concentration of binding molecules is added to the chamber. In some cases, the unfolding force of the nucleic acid that varies according to the concentration of the binding molecule is calculated for each concentration of the binding molecule.

[0138] As disclosed herein, a device can include a plurality of features. For example, a device can include at least 100, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 or more than 100000 features. Thus, a single binding experiment can be performed on a large number of single molecules simultaneously.

[0139] As disclosed herein, feature means a binding moiety at the surface of a flow cell, such moieties being covalently or non-covalently attached to the surface. These features allow RNA molecules to be anchored at the surface of the flow cell. These features can be proteins (e.g., antibodies or streptavidin) or oligonucleotides (similar to microarrays). The covalent attachment of the binding moiety at the surface of the flow cell can be performed by a technician, and includes esterification or click chemistry. In the case of using oligonucleotides, for all features, it can be the same sequence or different sequences. In some cases, different sequences with compositional features may be advantageous for anchoring different RNA molecules at specific locations in the flow cell. This allows for rapid mapping of different structures in the flow cell. The sequence of the RNA allowed to be anchored on the surface can be changed accordingly.

[0140] Exemplary embodiments

[0141] Embodiment 1 is provided herein, wherein embodiment 1 includes a method for screening binding or non-binding molecules of a nucleic acid molecule, the method comprising:

[0142] a. connecting a plurality of nucleic acid molecules to a plurality of beads via one end of the nucleic acid molecule;

[0143] b. connecting a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules;

[0144] c. contacting the chamber with a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber;

[0145] d. approaching a magnet to move the plurality of beads along the axis of the chamber relative to the bottom surface, and measuring changes in the positions of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface;

[0146] e. determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; and

[0147] f. selecting members of the library of binding molecules that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule.

[0148] Embodiment 2 is provided herein, wherein embodiment 2 includes the method according to embodiment 1, and the method also includes selecting members of the binding molecule library that do not produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule.

[0149] Embodiment 3 is provided herein, wherein embodiment 3 includes a method for identifying chemical labels of the best binders in a binding molecule library of nucleic acid molecules, the method comprising:

[0150] a. connecting a plurality of the nucleic acid molecules to a plurality of beads via the first end of the nucleic acid molecule;

[0151] b. connecting a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules;

[0152] c. contacting the chamber with a library of binding molecules, wherein each member of the library of binding molecules is contacted with a single nucleic acid molecule;

[0153] d. approaching a magnet to move the plurality of beads along the axis of the chamber relative to the bottom surface, and measuring changes in the positions of the beads by a sensor of the device based on the movement of the beads relative to the bottom surface;

[0154] e. determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library;

[0155] f. selecting members of the library of binding molecules that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force for the nucleic acid molecule in the absence of the binding molecule; and

[0156] g. Modeling selected members of the binding molecule library to identify chemical tags of the best binders in the binding molecule library.

[0157] Provided herein is Embodiment 4, wherein Embodiment 4 comprises the method according to any one of the preceding Embodiments, wherein the plurality of features comprises at least 10,000 features.

[0158] Embodiment 5 is provided herein, wherein Embodiment 5 comprises a method according to any of the preceding embodiments, wherein the sensor of (d) comprises a camera CMOS capable of collecting photons reflected from the beads.

[0159] Embodiment 6 is provided herein, wherein Embodiment 6 comprises a method according to any one of the preceding embodiments, wherein the sensor of (d) comprises a CMOS capable of measuring impedance from the feature caused by movement of the bead, particularly up and down movement of the bead.

[0160] Provided herein is embodiment 7, wherein embodiment 7 comprises a method according to any one of the preceding embodiments, wherein the nucleic acid molecule comprises a DNA molecule, an RNA molecule, or a combination thereof.

[0161] Embodiment 8 is provided herein, wherein embodiment 8 comprises a method according to any one of the preceding embodiments, wherein the nucleic acid molecule is a DNA / RNA hybrid molecule.

[0162] Embodiment 9 is provided herein, wherein embodiment 9 comprises a method according to any one of the preceding embodiments, wherein the multiple nucleic acid molecules are the same or different nucleic acid molecules.

[0163] Embodiment 10 is provided herein, wherein embodiment 10 comprises a method according to any of the preceding embodiments, wherein the force of the magnet relative to the beads is constant or increases.

[0164] Provided herein is embodiment 11, wherein embodiment 11 comprises the method according to any one of the preceding embodiments, wherein the nucleic acid molecule is about 20 bp to about 350 bp.

[0165] Provided herein is embodiment 12, wherein embodiment 12 comprises the method according to any one of the preceding embodiments, wherein the nucleic acid molecule is about 20 bases to about 350 bases.

[0166] Provided herein is embodiment 13, wherein embodiment 13 comprises the method according to any one of the preceding embodiments, wherein the nucleic acid molecule is chemically synthesized. Provided herein is embodiment 14, wherein embodiment 14 comprises the method according to any one of the preceding embodiments, wherein the nucleic acid molecule is produced by in vitro transcription.

[0167] Provided herein is embodiment 15, wherein embodiment 15 comprises the method according to any one of the preceding embodiments, wherein the nucleic acid molecule is produced by polymerase chain reaction.

[0168] Provided herein is embodiment 16, wherein embodiment 16 comprises a method according to any one of the preceding embodiments, wherein the binding molecule is a small molecule or a polypeptide.

[0169] Provided herein is embodiment 17, wherein embodiment 17 comprises a method according to any one of the preceding embodiments, wherein the device is configured to resolve differences in unfolding force between members of the binding molecule library of about 0.1-fold to about 30-fold.

[0170] Embodiment 18 is provided herein, wherein Embodiment 18 comprises a method according to any of the preceding embodiments, wherein the connection of (a) comprises biotin-streptavidin binding, wherein the biotin is conjugated to one end of the nucleic acid and the streptavidin is conjugated to the bead.

[0171] Embodiment 19 is provided herein, wherein embodiment 19 comprises a method according to any of the preceding embodiments, wherein the connection of (b) comprises a DNA oligonucleotide covalently connected to the bottom of each feature, wherein the DNA oligonucleotide has complementarity with the second end of the nucleic acid molecule.

[0172] Embodiment 20 is provided herein, wherein embodiment 20 comprises a method for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the method comprising:

[0173] a. connecting a plurality of the nucleic acid molecules to a plurality of beads via the first end of the nucleic acid molecule;

[0174] b. connecting a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules;

[0175] c. contacting the chamber with increasing concentrations of the binding molecule;

[0176] d. applying a magnetic field sufficient to move each bead of the plurality of beads relative to the bottom surface of the chamber, and measuring a change in the position of the bead by a sensor of the device based on the movement of the bead relative to the bottom surface;

[0177] e. determining the unfolding force of the nucleic acid molecule according to the concentration variation of the binding molecule; and

[0178] f. calculating the binding kinetics between the binding molecule and the nucleic acid molecule based on the change in the unfolding force according to the amount of the binding molecule.

[0179] Embodiment 21 is provided herein, wherein embodiment 21 comprises a method according to any one of the preceding embodiments, wherein the unfolding force of the nucleic acid molecule that varies according to the concentration of the binding molecule is calculated simultaneously for each concentration of the binding molecule.

[0180] Provided herein is Embodiment 22, wherein Embodiment 22 comprises a method according to any one of the preceding embodiments, wherein the binding molecule has a dissociation constant (K) of about 0.01 nM to about 100 mM. D ).

[0181] Embodiment 23 is provided herein, wherein Embodiment 23 comprises a method for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the method comprising:

[0182] a. contacting the nucleic acid molecules attached to the fixed end of the chamber and the magnetic beads on the opposite end with increasing concentrations of the binding molecules; and

[0183] b. Determining the unfolding force of the nucleic acid according to the concentration of the binding molecule; wherein the nucleic acid molecule is selected from the group consisting of: HIV-1TAR, preQ1 riboswitch, pre-miR-21, MYC, APOC3, DM1, C9orf72, MAPT and FMR1.

[0184] Provided herein is embodiment 24, wherein embodiment 24 comprises a method according to any one of the preceding embodiments, wherein the binding molecule is a small chemical molecule. Provided herein is embodiment 25, wherein embodiment 25 comprises a method according to any one of the preceding embodiments, wherein the binding molecule is a polypeptide.

[0185] Provided herein is Embodiment 26, wherein Embodiment 26 comprises a method according to any one of the preceding Embodiments, wherein the binding molecule has a dissociation constant (Kd) of about 0.01 nM to about 100 mM.

[0186] Embodiment 27 is provided herein, wherein Embodiment 27 comprises a method according to any of the preceding embodiments, wherein the chamber is disposed in a device configured to measure displacement of beads of the nucleic acid as a function of concentration of binding molecules.

[0187] Embodiment 28 is provided herein, wherein Embodiment 28 comprises a method according to any of the preceding embodiments, wherein the device comprises a plurality of features.

[0188] Embodiment 29 is provided herein, wherein embodiment 29 comprises a method according to any of the preceding embodiments, wherein a plurality of nucleic acid molecules are anchored to the bottom of the plurality of features, wherein each feature of the chamber comprises a single nucleic acid molecule sequence from the plurality of nucleic acid molecule sequences.

[0189] Embodiment 30 is provided herein, wherein embodiment 30 comprises a method according to any one of the preceding embodiments, wherein a single concentration of the binding molecule is added to the chamber, wherein the unfolding force of the nucleic acid that varies according to the concentration of the binding molecule is calculated for each concentration of the binding molecule. Provided herein is Embodiment 31, wherein Embodiment 31 comprises a method according to any of the preceding Embodiments, wherein the plurality of features comprises at least 10,000 features.

[0190] Provided herein is embodiment 32, wherein embodiment 32 comprises a method according to any one of the preceding embodiments, wherein the nucleic acid molecule comprises HIV-1 TAR, preQ1 riboswitch, pre-miR-2 or DM1 or a viral nucleic acid.

[0191] Provided herein is embodiment 33, wherein embodiment 33 comprises a method according to any one of the preceding embodiments, wherein the nucleic acid molecule is a preQ1 riboswitch.

[0192] Provided herein is embodiment 34, wherein embodiment 34 comprises the method according to any of the preceding embodiments, wherein relative to the wild-type preQ1 riboswitch, the preQ1 riboswitch comprises a mutation selected from the group consisting of: C15U, U7C, A10G, and U6C.

[0193] Embodiment 35 is provided herein, wherein Embodiment 35 comprises a system for practicing the method according to any of the preceding embodiments.

[0194] Embodiment 36 is provided herein, wherein embodiment 36 comprises a kit and container comprising a system for practicing any of the methods according to any of the preceding embodiments.

[0195] Embodiment 37 is provided herein, wherein Embodiment 37 comprises an apparatus comprising a system for practicing any of the methods according to any of the preceding embodiments.

[0196] Embodiment 38 is provided herein, wherein embodiment 38 comprises a system for screening binding molecules of nucleic acid molecules, the system comprising:

[0197] a. connecting a plurality of nucleic acid molecules to a plurality of beads via one end of the nucleic acid molecule;

[0198] b. a plurality of nucleic acid molecules connected to a plurality of features of a device via a second end of the nucleic acid molecule;

[0199] c. a chamber having a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber;

[0200] d. a magnet that moves the plurality of beads along the axis of the chamber relative to the bottom surface; and

[0201] e. A sensor that measures changes in the position of a bead of the device based on movement of the bead relative to the bottom surface.

[0202] Embodiment 39 is provided herein, wherein embodiment 39 comprises a system for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the system comprising:

[0203] a. connecting a plurality of nucleic acid molecules to a plurality of beads via one end of the nucleic acid molecule;

[0204] b. a plurality of nucleic acid molecules connected to a plurality of features of a device via a second end of the nucleic acid molecule;

[0205] c. a chamber having a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber;

[0206] d. a magnet that moves the plurality of beads along the axis of the chamber relative to the bottom surface; and

[0207] e. A sensor that measures changes in the position of a bead of the device based on movement of the bead relative to the bottom surface.

[0208] Embodiment 40 is provided herein, wherein embodiment 40 comprises a kit comprising:

[0209] a. a nucleic acid molecule or a plurality of the same or different nucleic acid molecules comprising a targeting sequence at one end and a biotin moiety at the other end;

[0210] b. a DNA handle comprising a first reactive group, wherein the DNA handle is capable of hybridizing to the directional sequence;

[0211] c. a feature of the device comprising a second reactive group capable of chemically reacting with the first reactive group; and

[0212] d. A bead or beads comprising a streptavidin moiety capable of binding to said biotin moiety.

[0213] Embodiment 41 is provided herein, wherein embodiment 41 comprises the kit according to the previous embodiment, further comprising a library of binding molecules.

[0214] Embodiment 42 is provided herein, wherein embodiment 42 comprises a kit according to any one of the preceding embodiments, wherein the binding molecule library comprises a small molecule library, a protein library, an antibody library, an aptamer library, advantageously comprising a library of binding molecules.

[0215] Provided herein is embodiment 43, wherein embodiment 43 comprises a kit according to any of the preceding embodiments, further comprising a buffer comprising a divalent cation in the range of 1 μM to 1 mM.

[0216] Provided herein is embodiment 44, wherein embodiment 44 comprises a kit according to any one of the preceding embodiments, wherein the divalent cation comprises Mg 2+ , Mn 2+ 、Zn 2+ , Fe 2+ , Cu 2+or Cd 2+ .

[0217] Provided herein is embodiment 45, wherein embodiment 45 comprises a kit according to any one of the preceding embodiments, wherein the chemical reaction is an esterification reaction.

[0218] Provided herein is embodiment 46, wherein embodiment 46 comprises a kit according to any one of the preceding embodiments, wherein the chemical reaction is click chemistry.

[0219] Example

[0220] In order to better understand the present disclosure and its many advantages, the following examples are given by way of illustration and not to limit the scope of the present disclosure.

[0221] Example 1: Measuring the binding affinity of different ligands to HIV-1 TAR

[0222] A 59-nucleotide HIV-TAR RNA flanking the RNA / DNA duplex was attached to paramagnetic beads on one side and to a flow cell on the other. The ramp experiment showed characteristic traces of unfolding and refolding forces with a hysteresis in between ( FIG. 2B to FIG. 2E Addition of 10 mM arginine amide increased the unfolding and refolding forces ( Figure 2B ), demonstrating that arginine amide binding increases the stability of HIV-TAR RNA molecules. Addition of increasing concentrations of arginine amide increased the unfolding force, and a K of 5.43 mM was calculated by fitting the normalized force at the maximum unfolding distribution of each bead. D ( Figure 2B ). This demonstrates that the method described herein is suitable for studying the binding affinity of small molecules to RNA secondary structures.

[0223] To investigate whether the platform could discriminate between different KDs, three additional small molecules known to bind to the HIV-1 TAR structure with different binding affinities were used ( FIG. 2C to FIG. 2E ). Netilmicin is a selective binder to HIV-1 TAR with a KD of 1.4 μM and a Ki of 14 μM. In fact, increasing the concentration of netilmicin resulted in an increase in the unfolding force, and the calculated KD was about 13.4 μM ( Figure 2C ). The nonspecific aminoglycoside antibiotic kanamycin can also bind to pyrene-labeled HIV-1 TAR with a KD of 107 μM. Addition of kanamycin leads to an increase in the unfolding force ( Figure 2D ). Above 100 μM kanamycin, there is a broadening of the force distribution ( Figure 2DFitting the force at the maximum normalized distribution showed an increase in the unfolding force when kanamycin was present, with a calculated KD of 10 μM. 3-amino-6-methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide, designated compound 4, was also tested, which is a specific binder to HIV-1 TAR with a KD of 2.4 μM. Addition of compound 4 resulted in a decrease in the unfolding force ( Figure 2E ), which may be the result of the non-classical binding of this molecule to the lower protrusion, thereby destabilizing the HIV-1 TAR structure. Fitting the change in the unfolding force gave a KD of 2.99 μM.

[0224] In summary, the platform described herein is used to measure the binding affinity of different small molecules. According to the ramp experiment, if the ligand binding causes a change in RNA structural stability, the system detects the change in unfolding and refolding power. The difference in binding affinity (KD) of different small molecules can be calculated, making the platform suitable for screening drug libraries for specific RNA targets.

[0225] Example 2 - Binding of ligands to the PreQ1 riboswitch

[0226] This example demonstrates ligand binding to the Bsu PreQ1 riboswitch. The Bsu riboswitch exhibits a major population of a 'prefolded' state, in which the 3' tail transiently interacts with the PI-LI stem-loop ( Figure 3A Given the conformational rearrangement upon ligand binding to the Bsu preQ1 riboswitch, we observed changes between the unfolded and folded states under constant force experiments.

[0227] To study the molecular dynamics of the preQ1 riboswitch using the platform described herein, a wild-type (WT) Bsu riboswitch was attached to a flow cell as described in Example 1. A stepped constant force experiment was then performed, in which the molecule was held at a constant force for a given amount of time ( ) before increasing the force in a stepwise manner from lower to higher forces. Figure 3B ). The z position of the bead is recorded, and under forces closer to equilibrium, the molecule transitions between folded and unfolded states ( Figure 3B The force was kept constant for five minutes at equilibrium (where the molecule spends the same amount of time in the unfolded state as in the folded state), and various concentrations of preQ1 ligand were tested. Binding of the ligand to the structure was monitored in real time at the single molecule level ( Figure 3C The arrows are shown in the representative binding analysis curves of ). Bindings that caused the structure to remain in the folded state for more than 3 seconds were recorded. k was calculated by dividing the number of events observed when the ligand bound to the RNA structure by the time of the experiment at a given concentration of ligand. onWhen the RNA remains folded for more than 3.3 seconds, it is considered to be bound to the ligand (here, the preQ1 ligand). By testing different concentrations, the fen( Figure 3D ). off is the inverse of the length of time that the RNA structure is bound by the ligand. Therefore, the duration when the ligand leaves the RNA structure (star) is observed because the RNA structure quickly transitions back between the folded and unfolded states. The duration of this folded state allows the measurement of the k of the ligand. off ( Figure 3E ). on depends on the ligand concentration, while k off For each force, a hidden Markov model was used to fit the raw data to two different states ( Figure 3B ), and the fraction of time each molecule spends in the folded state relative to the time of each force step is calculated. This fraction is plotted against the force of each step, and the equilibrium force (inflection point) for each molecule is determined when the molecule spends 50% of its time in each state ( Figure 3F The half-width of the force can also be determined by calculating 1 / e (e = Euler's number) on either side of the inflection point, which indicates the range of forces over which the molecule is bistable and transitions from the folded and unfolded states ( Figure 3F The transition frequency was calculated for each force step recorded and the normalized force was again plotted, corresponding to the equilibrium force for each bead under control conditions ( Figure 3G ).

[0228] To determine the Mg 2+ Whether the presence of Mg has an effect on folding confirmation, different concentrations of Mg 2+ In practice, 1 mM Mg was added to each flow cell. 2+ Increases the unfolding equilibrium of the same molecule without affecting the range ( Figure 3J ), and the transition frequency decreases when the force is balanced ( Figure 3K ), indicating that in the absence of ligands Mg 2+ Stabilizing effect on preQ1 RNA structure. Addition of 1 μM preQ1 ligand (above the value for preQ1 KD) also increased the unfolding equilibrium and decreased the transition frequency, albeit to a lesser extent ( Figure 3J and Figure 3K ). Addition of both ligand and cation showed additive effects ( Figure 3J and Figure 3K ), indicating that the two molecules bind to the RNA structure at different locations. For subsequent experiments, 1 mM Mg was added to all buffers. 2+ .

[0229] To investigate the effect of preQ1 ligand binding to the riboswitch, constant force step experiments were performed with increasing concentrations of preQ1 ligand ( Figure 3B Quantification showed that increasing concentrations of preQ1 increased the equilibrium force of the riboswitch ( Figure 3F ) and reduce the switching frequency ( Figure 3F The KD (162 nM) was calculated by fitting the equilibrium forces at each concentration and compared with the results obtained by smFRET (134 + / - 48 nM).

[0230] Constant force experiments were performed at 9.6 pN (the median of the fitted equilibrium forces of all beads under control conditions) for extended periods of time (30 min-60 min, Figure 3G The molecules were detected to transition between folded and unfolded states, and two distinct dynamics were observed: (i) rapid transitions between folded and unfolded states (~8 transitions-10 transitions / s) and (ii) molecules remaining folded for longer periods of time ( Figure 3G ). The probability of the prolonged folded state increases with the preQ1 concentration. Ligand binding is defined when the molecule remains folded for more than 3.3 seconds, while when the ligand is not bound, the molecule rapidly transitions between the two states. The average time per ligand binding event (folded state) was calculated for each concentration, and Koff (once per average time) was shown to be unaffected by the ligand concentration ( Figure 3H ), where the value is 0.11s -1 For different concentrations, the ligation rate was calculated by dividing the number of binding events by the time the molecule spends in the folded state without ligation (during the fast transition). Therefore, k calculated by fitting the ligation rate on 0.0015s -1 nM -1 ( Figure 3H ). Therefore, through k off / k on The KD at 9.6 pN was calculated to be 73 nM.

[0231] In summary, the platform described in this paper demonstrates the ability to study the kinetics of small molecule binding to RNA structures by using constant force experiments. The rates of association and dissociation can be derived from this type of experiment, which gives more insight into the binding kinetics of the ligand. Therefore, this platform is also suitable for studying detailed single-molecule folding dynamics.

[0232] Example 3 - Effect of preQ1 mutation on ligand binding

[0233] This example demonstrates that the platform described herein can address the effects of nucleic acid scaffold mutations on ligand binding. The preQ1 riboswitch has several point substitutions that alter the affinity for the preQ1 ligand ( Figure 4A To demonstrate that the platform described herein can be used to report changes in binding affinity using different RNA substrates, we selected four mutants that increased or decreased binding affinity (Table 5).

[0234] Table 5: Effect of PreQ1 mutations on binding affinity

[0235]

[0236] WT preQ1 riboswitch was used as reference ( Figure 4B Using slope analysis, the unfolding force distribution shows a broadening of the distribution ( Figure 4B The peak near the reference force decreases, while the fraction of cycles with higher unfolding forces increases. The higher force cycles may represent the binding of the ligand and the formation of pseudoknots. The fraction of cycles with an unfolding force greater than the 95% confidence interval for each bead under control conditions is calculated ( Figure 4B Fitting the high force fraction provided a calculated KD of 87.8 + / - 9.02 nM ( Figure 4B ). This is similar to the KD calculated from the constant force experiment at 9.6 pN in Example 2.

[0237] After establishing the reference point, four different mutants were used to study the effect on preQ1 binding ( Figures 4C to 4G Ramp analysis of the C15U mutant showed similar behavior to the WT, with an increase in the fraction of high forces following preQ1 binding ( Figure 4C Fitting the high force fraction showed a KD of 5.35 + / - 1.75 nM ( Figure 4C The double mutants U7C and C15U also showed an increase in KD ( Figure 4D , 17.72+ / -5.9nM), with a higher KD than the C15U mutant. This difference may be due to the Mg used 2+ Nevertheless, the platform was able to discriminate a 3-fold difference in KD, which is less pronounced in platforms such as SPR or fluorescence titration.

[0238] The third mutant, A10G, showed reduced binding affinity for preQ1. In fact, the effect on the unfolding force was observed in the micromolar range rather than the nanomolar range ( Figure 4E The unfolding force distribution also changes. With this mutant, the entire distribution is shifted toward higher forces, instead of having two distinct fractions ( Figure 4E). This suggests that the ligand may slightly stabilize the existing structure, but may not allow the formation of a pseudoknot that is more difficult to unfold. The maximum unfolding force density for each bead was used to fit the KD (as in Example 1), and the KD was 34.67+ / -4.56 μM, which is about 400 times higher than WT ( Figure 4E ). Constant force step experiments also reveal equilibrium force changes in the micromolar range ( Figure 4F ).

[0239] The final mutant, U6C, abolished ligand binding. The unfolding force profile was visualized as a broadening of the peak, but the maximum peak was unchanged ( Figure 4G ). Fitting the maximum density ( Figure 4G ) and high force density yielded no KD values, indicating that the U6C mutant abolished binding of the preQ1 ligand.

[0240] In summary, the platform described herein is able to detect binding affinities ranging from 3-fold to 400-fold differences. Therefore, the platform can be used to study the effects of sequence mutations on small molecule binding. It can also be used to determine the specificity of a single small molecule to a library of RNA molecules.

[0241] Example 4 - Measuring the interaction between pre-miR-21 and Dicer protein.

[0242] Pre-miR-21 and pre-miR-21-bp mutants are used as substrates to measure the effect of Dicer on folding dynamics. Dicer is a double-stranded specific ribonuclease that binds pre-miRNA and cuts them to produce mature miRNA. Adding 20nM of full-length Dicer without magnesium causes the binding of protein to structure without further processing of the structure. The binding of Dicer to pre-miR-21 structure occurs as the unfolding force increases. There is no binding to the mutant form of pre-miR-21-bp (the mutant introduces a mutation that eliminates the protrusion required for Dicer binding). Therefore, the platform described herein can be used to detect the interaction between RNA scaffold and RNA binding protein.

[0243] In summary, the platform described herein allows the study of not only the interaction between nucleic acids and small molecules, but also the interaction between proteins and nucleic acids. The next step is to use binding proteins with secondary structures and observe whether the presence of small molecules can affect this interaction. Therefore, this platform can be used to study inhibition or competition between different molecules.

[0244] Although exemplary embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of the present disclosure, and methods and structures within the scope of these claims and their equivalents are thereby covered.

Claims

1. A method for screening binding or non-binding molecules of nucleic acid molecules, the method comprising: (a) connecting a plurality of nucleic acid molecules to a plurality of beads via one end of the nucleic acid molecules; (b) attaching a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules; (c) contacting the chamber with a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber; (d) approaching a magnet to move the plurality of beads relative to a bottom surface along an axis of the chamber, and measuring changes in the positions of the beads via a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; as well as (f) selecting members of the library of binding molecules that produce an unfolding force of the nucleic acid molecule, The unfolding force is greater or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule.

2. The method according to claim 1 further comprises selecting members of the binding molecule library that do not produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule.

3. A method for identifying chemically labeled optimal binders in a binding molecule library of nucleic acid molecules, the method comprising: (a) attaching a plurality of nucleic acid molecules to a plurality of beads via the first ends of the nucleic acid molecules; (b) attaching a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules; (c) contacting the chamber with a library of binding molecules, wherein each member of the library of binding molecules is contacted with a single nucleic acid molecule; (d) approaching a magnet to move the plurality of beads relative to a bottom surface along an axis of the chamber, and measuring changes in the positions of the beads via a sensor of the device based on the movement of the beads relative to the bottom surface; (e) determining the unfolding force of the nucleic acid molecule in the presence of each member of the binding molecule library; (f) selecting members of the library of binding molecules that produce an unfolding force for the nucleic acid molecule that is greater than or less than the unfolding force of the nucleic acid molecule in the absence of the binding molecule; as well as (g) Modeling selected members of the binding molecule library to identify the chemical signature of the best binder in the binding molecule library.

4. The method of claim 1 or 3, wherein the plurality of features comprises at least 10,000 features.

5. The method of claim 1 or 3, wherein the sensor of (d) comprises a camera CMOS capable of collecting photons reflected from the beads.

6. The method of claim 1 or 3, wherein the sensor of (d) comprises a CMOS capable of measuring impedance from the feature caused by the bead moving up and down.

7. The method according to claim 1 or 3, wherein the nucleic acid molecule is a DNA molecule.

8. The method of claim 1 or 3, wherein the nucleic acid molecule is an RNA molecule.

9. The method according to claim 1 or 3, wherein the nucleic acid molecule is a DNA / RNA hybrid molecule.

10. The method of claim 1 or 3, wherein the plurality of nucleic acid molecules are identical nucleic acid molecules.

11. The method of claim 1 or 3, wherein the plurality of nucleic acid molecules are different nucleic acid molecules.

12. The method of claim 1 or 3, wherein the force of the magnet relative to the beads is constant.

13. The method of claim 1 or 3, wherein the force of the magnet relative to the beads is increased.

14. The method of claim 1 or 3, wherein the nucleic acid molecule is about 20 bp to about 350 bp.

15. The method of claim 1 or 3, wherein the nucleic acid molecule is about 20 bases to about 350 bases.

16. The method of claim 1 or 3, wherein the nucleic acid molecule is chemically synthesized.

17. The method of claim 1 or 3, wherein the nucleic acid molecule is produced by in vitro transcription.

18. The method of claim 1 or 3, wherein the nucleic acid molecule is produced by polymerase chain reaction.

19. The method of claim 1 or 3, wherein the binding molecule is a small molecule.

20. The method of claim 1 or 3, wherein the binding molecule is a polypeptide.

21. The method of claim 1 or 3, wherein the device is configured to resolve differences in unfolding force between members of the binding molecule library of about 0.1-fold to about 30-fold.

22. The method of claim 1 or 3, wherein the attachment of (a) comprises biotin-streptavidin binding, wherein the biotin is conjugated to one end of the nucleic acid and the streptavidin is conjugated to the bead.

23. The method of claim 1 or 3, wherein the linking of (b) comprises a DNA oligonucleotide covalently linked to the base of each feature, wherein the DNA oligonucleotide has complementarity with the second end of the nucleic acid molecule.

24. A method for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the method comprising: (a) attaching a plurality of nucleic acid molecules to a plurality of beads via the first ends of the nucleic acid molecules; (b) attaching a plurality of nucleic acid molecules to a plurality of features of a device via the second ends of the nucleic acid molecules; (c) contacting the chamber with increasing concentrations of the binding molecule; (d) applying a magnetic field sufficient to move each bead of the plurality of beads relative to the bottom surface of the chamber, and measuring a change in the position of the bead via a sensor of the device based on the movement of the bead relative to the bottom surface; (e) measuring the unfolding force of the nucleic acid molecule according to the concentration of the binding molecule; as well as (f) calculating the binding kinetics between the binding molecule and the nucleic acid molecule based on the change in the unfolding force according to the change in the amount of the binding molecule.

25. The method according to claim 24, wherein the unfolding force of the nucleic acid molecule that varies according to the concentration of the binding molecule is calculated simultaneously for each concentration of the binding molecule.

26. The method of claim 24 or 25, wherein the binding molecule has a dissociation constant (KD) of about 0.01 nM to about 100 mM.

27. A method for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the method comprising: (a) contacting the nucleic acid molecules attached to the fixed end of the chamber and the magnetic beads on the opposite end with increasing concentrations of the binding molecules; as well as (b) measuring the unfolding force of the nucleic acid according to the concentration of the binding molecule; in The nucleic acid molecule is selected from the group consisting of HIV-1 TAR, preQ1 riboswitch, pre-miR-21, MYC, APOC3, DM1, C9orf72, MAPT and FMRI.

28. The method of claim 27, wherein the binding molecule is a small chemical molecule.

29. The method of claim 27, wherein the binding molecule is a polypeptide.

30. The method of claim 27, wherein the binding molecule has a dissociation constant (KD) of about 0.01 nM to about 100 mM.

31. The method of claim 27, wherein the chamber is disposed in a device configured to measure displacement of beads of the nucleic acid as a function of the concentration of the binding molecule.

32. The method of claim 31 , wherein the device comprises a plurality of features.

33. The method of claim 32, wherein a plurality of nucleic acid molecules are anchored to the bottom of the plurality of features, wherein each feature of the chamber comprises a single nucleic acid molecule sequence from the plurality of nucleic acid molecule sequences.

34. The method of claim 33, wherein a single concentration of the binding molecule is added to the chamber, wherein the unfolding force of the nucleic acid as a function of the concentration of the binding molecule is calculated for each concentration of the binding molecule.

35. The method of any one of claims 32, wherein the plurality of features comprises at least 10,000 features.

36. The method of claim 27, wherein the nucleic acid molecule is HIV-1 TAR.

37. The method of claim 27, wherein the nucleic acid molecule is a preQ1 riboswitch.

38. The method of claim 37, wherein the preQ1 riboswitch comprises a mutation selected from the group consisting of: C15U, U7C, A10G, and U6C relative to a wild-type preQ1 riboswitch.

39. The method of claim 27, wherein the nucleic acid molecule is pre-miR-21.

40. The method of claim 27, wherein the nucleic acid molecule is DM1.

41. A system for practicing the method of claim 1, 3, 24 or 27.

42. A kit and container comprising a system for practicing the method of claim 1, 3, 24 or 27.

43. An apparatus comprising a system for practicing the method of claim 1, 3, 24 or 27.

44. A system for screening binding molecules for nucleic acid molecules, the system comprising: (a) a plurality of nucleic acid molecules attached to a plurality of beads via one end of the nucleic acid molecules; (b) a plurality of nucleic acid molecules attached to a plurality of features of a device via second ends of the nucleic acid molecules; (c) a chamber having a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber; (d) a magnet that moves the plurality of beads along the axis of the chamber relative to the bottom surface; as well as (e) a sensor for measuring changes in the position of a bead of the device based on movement of the bead relative to the bottom surface.

45. A system for determining the binding kinetics of a binding molecule to a nucleic acid molecule, the system comprising: (a) a plurality of nucleic acid molecules attached to a plurality of beads via one end of the nucleic acid molecules; (b) a plurality of nucleic acid molecules attached to a plurality of features of a device via second ends of the nucleic acid molecules; (c) a chamber having a library of binding molecules, wherein each member of the library of binding molecules is contacted with a plurality of single nucleic acid molecules in a single chamber; (d) a magnet that moves the plurality of beads along the axis of the chamber relative to the bottom surface; as well as (e) a sensor for measuring changes in the position of a bead of the device based on movement of the bead relative to the bottom surface.

46. ​​A kit, comprising: (a) a nucleic acid molecule or a plurality of the same or different nucleic acid molecules comprising a targeting sequence at one end and a biotin moiety at the other end; (b) a DNA handle comprising a first reactive group, wherein the DNA handle is capable of hybridizing to the targeting sequence; (c) a feature of the device comprising a second reactive group capable of chemically reacting with the first reactive group; and (d) a bead or beads comprising a streptavidin moiety capable of binding to said biotin moiety.

47. The kit of claim 46, further comprising a library of binding molecules.

48. The kit according to claim 47, wherein the binding molecule library comprises a small molecule library, a protein library, an antibody library, an aptamer library, advantageously a library of binding molecules.

49. The kit of claim 46, further comprising a buffer comprising divalent cations in the range of 1 μM to 1 mM.

50. The kit of claim 49, wherein the divalent cations include Mg 2+ , Mn 2+ 、Zn 2+ , Fe 2+ , Cu 2+ or Cd 2+ .

51. The kit of claim 46, wherein the chemical reaction is an esterification reaction.

52. The kit of claim 46, wherein the chemical reaction is click chemistry.

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