Apparatus for determining molecular interactions

Abstract

Disclosed herein are methods of determining binding interactions between two or more candidate molecules. Also disclosed herein are methods of determining binding kinetics of two or more candidate molecules. Also disclosed herein are methods for screening two candidate molecules for having a binding interaction with each other.

Description

[0001] Cross-reference applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 520,896, filed August 21, 2023, the entire contents of which are incorporated herein by reference.

[0003] Incorporate into the sequence list by reference

[0004] This application contains a sequence list, which is submitted with the application. The sequence list is named 201326-702601_PCT_SL.xml, created on August 19, 2024, and is 76,037 bytes in size, and is incorporated herein by reference in its entirety. Background Technology

[0005] Candidates that can bind to target molecules can be identified through high-throughput screening. High-throughput screening allows the use of automated equipment to test thousands to millions of molecules. To identify candidates, one strategy consists of first identifying low-quality hits based on a fragment library consisting of simple scaffold chemical structures, and then affine the selection, for example, by modifying high-quality hits to design candidates. In parallel, various doses of the selected candidates can also be tested to quantitatively analyze molecular behavior and potentially predict their activity.

[0006] Attempts have been made to explore these molecular interactions, which typically occur at low forces, using complex systems composed of fluorescence and dsDNA leaching. However, these systems are difficult to position properly and cannot be extended to a wide range of interactions. Furthermore, modifying proteins or molecules with fluorophores often introduces the risk of them becoming inactive or their interactions with other molecules being interfered with. The large number of operations required to attach binding molecules to the system makes it impossible to generalize the system and achieve high throughput for testing different interactions in parallel.

[0007] To improve the selection of these potential candidates and / or obtain more meaningful results, several methods have been developed, such as the highly sensitive optical method "surface plasmon resonance (SPR)." SPR occurs when polarized light strikes a conductive surface at the interface between two media. This generates an electron charge density wave called a plasmon, which proportionally reduces the intensity of reflected light at a specific angle (called the resonance angle) relative to the mass on the sensor surface. Therefore, this technique allows for the real-time detection of molecular interactions. However, this still relies solely on the binding between molecules. Attempts have been made to identify methods to improve the accuracy of candidate selection, but these methods remain 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] This document provides a nucleic acid scaffold for determining binding interactions between a first candidate molecule and a second candidate molecule. In some embodiments, the nucleic acid scaffold comprises: (a) a continuous polynucleotide sequence comprising: (i) a first end attached to a bead, wherein the first end comprises a first molecule-binding sequence; (ii) a second end attached to a bottom surface of the device, wherein the second end comprises a second molecule-binding sequence; and (iii) an intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises: (I) a first lead-forming sequence comprising a barcode; and (II) a second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence and the second lead-forming sequence are complementary. The sequence includes (a) hybridization of the lead-forming sequence and (b) a loop connecting the first lead-forming sequence and the second lead-forming sequence; (c) a first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and (d) a second spacer polynucleotide having a polynucleotide sequence complementary to the second lead-forming sequence, wherein the first spacer polynucleotide and the second spacer polynucleotide hybridize with the intermediate portion, and wherein the nucleic acid scaffold requires a force applied to the bead along an axis perpendicular to the bottom surface of the device to unfold the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold. In some embodiments, the force may be from 0.1 pN to 10 pN. In some embodiments, the barcode sequence comprises one or more modified nucleotides. In some embodiments, the intermediate portion further includes a first linker sequence and a second linker sequence, wherein the first linker sequence is located between the first molecule-binding sequence and the first lead-forming sequence, and wherein the second linker sequence is located between the second molecule-binding sequence and the second lead-forming sequence, and wherein the first linker sequence and the second linker sequence are not complementary to each other. In some embodiments, the first spacer polynucleotide and the second spacer polynucleotide are linked to each other by a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop. In some embodiments, the first molecule-binding sequence and the second molecule-binding sequence each independently comprise a small hairpin nucleic acid having a size ranging from 5 to 100 bases.

[0009] This document also provides screening nucleic acid scaffolds. In some embodiments, the screening nucleic acid scaffold comprises: (a) any of the nucleic acid scaffolds described herein; (b) a first candidate molecule ligated to a first gel sequence, wherein the first gel sequence hybridizes with the first molecule binding sequence; and (c) a second candidate molecule ligated to a second gel sequence, wherein the second gel sequence hybridizes with the second molecule binding sequence.

[0010] This document also provides an apparatus comprising: (a) a chamber disposed within the apparatus, wherein the chamber includes a bottom surface capable of securing a nucleic acid scaffold; (b) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and (c) any of the nucleic acid scaffolds described herein or a screening nucleic acid scaffold described herein.

[0011] This document also provides a method for determining the binding interaction between a first candidate molecule and a second candidate molecule. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of a chamber of the device; (b) providing a device comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface capable of fixing the nucleic acid scaffold, and (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence and the second candidate molecule are linked to a first gel sequence. The first molecule binding sequence is complementary, and the second gel sequence is complementary to the second molecule binding sequence; (d) the reference elongation length of the nucleic acid scaffold in response to force in the absence of the first candidate molecule and the second candidate molecule is determined in real time by: (i) applying a force of 0.1 pN to 50 pN to the beads attached to the nucleic acid scaffold along the axis perpendicular to the bottom surface of the device via the force application mechanism, wherein the nucleic acid scaffold is configured to unfold in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and (ii) via a transmission The sensor measures the change in the position of the bead along the axis to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule; (e) the nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are positioned ... The second candidate molecules are adjacent to each other; (f) by repeating (d), the elongation length of the screening nucleic acid scaffold in response to the same force applied in the absence of the first candidate molecule and the second candidate molecule is determined; and (g) a difference is calculated, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and the reference elongation length, wherein a non-zero difference indicates that the binding interaction exists between the first candidate molecule and the second molecule under the applied force, and wherein a zero difference indicates that the binding interaction does not exist between the first candidate molecule and the second candidate molecule under the applied force.In some embodiments, the method further includes determining the binding energy (enthalpy, entropy, and ΔG) of the binding interaction between the first candidate molecule and the second candidate molecule by: (h) after (g), removing the force applied to the bead by the force-applying mechanism, thereby causing relaxation of the screening nucleic acid scaffold; and (i) repeating (f) to (h) and calculating the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length as a function of time. In some embodiments, the method further includes: (i) determining the Kon of the binding of the second molecule to the first candidate molecule, wherein the Kon is calculated based on the number of cycles of repeating (f) to (h) that result in the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length. In some embodiments, the method further includes: (h) determining the Koff of the binding of the second molecule to the first candidate molecule, wherein the Koff is calculated based on the length of time during each cycle of repeating (f) to (h) that the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length exists.

[0012] This document also provides a method for determining binding interactions between a first candidate molecule, a second candidate molecule, and a third candidate molecule. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of a chamber of the device; (b) providing a device comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface; and (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and (c) providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule. (d) Determining a reference elongation length of the nucleic acid scaffold in the absence of the first and second candidate molecules by: (i) applying a force of 0.1 pN to 50 pN along an axis perpendicular to the bottom surface of the chamber of the device via the force application mechanism to the beads attached to the nucleic acid scaffold, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis; and (ii) measuring the change in the position of the beads along the axis via a sensor, thereby determining the reference elongation length of the nucleic acid scaffold in the absence of the first and second candidate molecules. (e) Contacting the nucleic acid scaffold with the first candidate molecule and the second candidate molecule, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to each other; determining, by repeating (d), the force required to achieve an elongation length of the screening nucleic acid scaffold equal to the reference elongation length, wherein the required force is in the range of 0.1 pN to 50 pN; (e) Contacting the third candidate molecule with the screening nucleic acid scaffold; and (f) determining, by repeating (d), the force required in the presence of the third candidate molecule and in response to ( f) the elongation length of the screening nucleic acid scaffold under the same applied force; and g) calculating the difference, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule, wherein a non-zero difference indicates that there is a binding interaction between the third candidate molecule and the first candidate molecule and the second candidate molecule under the applied force, and wherein a zero difference indicates that there is no binding interaction between: (A) the third candidate molecule and the first candidate molecule, (B) the third candidate molecule and the second candidate molecule, or (C) the third candidate molecule under the applied force, and the first candidate molecule and the second candidate molecule.In some embodiments, the method further includes determining the binding energy (enthalpy, entropy, and ΔG) of the binding interaction between the first candidate molecule, the second candidate molecule, and the third candidate molecule, and optionally determining the binding kinetics thereby: (j) after (i), removing the force applied to the bead by the force-applying mechanism, thereby causing relaxation of the test screening nucleic acid scaffold; and (k) repeating (e) to (j) and calculating the difference in the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule as a function of time. In some embodiments, the method further includes: (j) determining the Kon of the binding of the third candidate molecule to the first and second molecules, wherein the Kon is calculated based on the number of cycles of repeating (h) to (j) that result in the difference in the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule. In some embodiments, the method further includes: (j) determining the Koff of the binding of the third candidate molecule to the first candidate molecule and the second candidate molecule, wherein the Koff is calculated based on the length of time during each cycle of repetitions (h) to (j) that the difference exists between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule.

[0013] This document also provides a method for screening binding interactions between first candidate molecules, second candidate molecules, and multiple third candidate molecules, the method comprising: (a) providing an apparatus comprising: (i) a chamber disposed within the apparatus, wherein the chamber includes a bottom surface; (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and (iii) a nucleic acid scaffold positioned along an axis perpendicular to the bottom surface of the chamber of the apparatus, wherein the nucleic acid scaffold comprises the nucleic acid scaffold described herein, wherein the nucleic acid scaffold is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the apparatus; (b) by The reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule is determined in the following manner: (iv) applying a force of 0.1 pN to 50 pN to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis; and (v) measuring the change in the position of the beads along the axis via a sensor, thereby determining the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the plurality of second candidate molecules; (c) connecting the nucleic acid scaffold to the first (d) Contact the first candidate molecule of the gel sequence to anchor the first candidate molecule to the nucleic acid scaffold; (d) Contact the nucleic acid scaffold with the second candidate molecule connected to the second gel sequence to anchor the second candidate molecule to the nucleic acid scaffold, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule of the screening nucleic acid scaffold are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other; (e) By repeating (d), determine the force required to achieve an elongation length of the screening nucleic acid scaffold that is the same as the reference elongation length, wherein the force is in the range of 0.1 pN to 50 pN; (f) Contact the plurality of third candidate molecules with the screening nucleic acid scaffold; (g) By repeating (b), In the presence of at least one of the plurality of third candidate molecules, the elongation length of the screening nucleic acid scaffold is determined in response to the same force applied in (e); and (h) the difference between the reference elongation length and the elongation length of the screening nucleic acid scaffold is calculated, wherein a non-zero difference indicates that there is a binding interaction between the third candidate molecule and the first and second candidate molecules under the applied force, and wherein a zero difference indicates that there is no binding interaction between: (A) the third candidate molecule and the first candidate molecule, (B) the third candidate molecule and the second candidate molecule, or (C) the third candidate molecule under the applied force, and the first and second candidate molecules.

[0014] This paper also provides a method for screening binding interactions between a first candidate molecule and multiple second candidate molecules.In some embodiments, the method includes: (a) providing an apparatus comprising: (i) a chamber disposed within the apparatus, wherein the chamber includes a bottom surface; (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and (iii) a plurality of nucleic acid scaffolds positioned along an axis perpendicular to the bottom surface of the chamber of the apparatus, wherein each of the plurality of nucleic acid scaffolds includes any of the nucleic acid scaffolds described herein, wherein each of the plurality of nucleic acid scaffolds is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the apparatus; (b) determining, in the absence of the first In the case of a candidate molecule and the plurality of second candidate molecules, the reference elongation length of each of the plurality of nucleic acid scaffolds is determined by: (i) applying a force of 0.1 pN to 50 pN along an axis perpendicular to the bottom surface of the chamber of the device via a force application mechanism to the bead attached to each of the plurality of nucleic acid scaffolds, wherein each of the plurality of nucleic acid scaffolds is configured to unfold in response to the applied force, thereby causing a change in the position of the bead attached to each of the plurality of nucleic acid scaffolds along the axis; and (ii) measuring the change in the position of the bead along the axis via a sensor to determine the situation in the absence of the first candidate molecule and the plurality of second candidate molecules. (c) Contacting the plurality of nucleic acid scaffolds with the first candidate molecule linked to the first gel sequence, thereby anchoring the first candidate molecule to each of the plurality of nucleic acid scaffolds, wherein the first gel sequence is complementary to the first molecule binding sequence; (d) Contacting the plurality of nucleic acid scaffolds with the plurality of second candidate molecules each linked to the second gel sequence, thereby anchoring one of the second candidate molecules to each of the plurality of nucleic acid scaffolds, wherein the second gel sequence is complementary to the second molecule binding sequence, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule of each of the plurality of screening nucleic acid scaffolds and the first candidate molecule of the first gel sequence are reference elongation lengths; Two candidate molecules are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other; (e) by repeating (d), the elongation length of each of the plurality of screening nucleic acid scaffolds in response to a force of 0.1 pN to 50 pN is determined; and (f) the difference between the reference elongation length and the elongation length of each of the plurality of screening nucleic acid scaffolds is calculated, wherein the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length indicates that the first candidate molecule and the second candidate molecule anchored to the screening nucleic acid have a binding interaction with each other under the applied force, and wherein the absence of the difference indicates that there is no binding interaction between the first candidate molecule and the second candidate molecule under the applied force.

[0015] This paper presents a method for screening binding interactions between first candidate molecules, second candidate molecules, and multiple third candidate molecules. In some embodiments, the method includes: (a) providing an apparatus comprising: (i) a chamber disposed within the apparatus, wherein the chamber includes a bottom surface; (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and (iii) a plurality of nucleic acid scaffolds positioned along an axis perpendicular to the bottom surface of the chamber of the apparatus, wherein each of the plurality of nucleic acid scaffolds includes any of the nucleic acid scaffolds described herein, wherein each of the plurality of nucleic acid scaffolds is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the apparatus; and (b) determining a reference elongation length of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the second candidate molecule by: (i) applying a force of 0.1 pN to 50 pN to the bead attached to each of the plurality of nucleic acid scaffolds via the force application mechanism along the axis perpendicular to the bottom surface of the chamber of the apparatus, wherein each of the plurality of nucleic acid scaffolds is The process involves (i) constructing a structure that unfolds in response to an applied force, resulting in a change in the position of the bead attached to each of the plurality of nucleic acid scaffolds along the axis, and (ii) measuring the change in the position of the bead along the axis via a sensor to determine a reference elongation length for each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules; (c) contacting the plurality of nucleic acid scaffolds with the first candidate molecule attached to a first gel sequence to anchor the first candidate molecule to each of the plurality of nucleic acid scaffolds; (d) contacting the plurality of nucleic acid scaffolds with the plurality of second candidate molecules respectively attached to a second gel sequence to anchor the second candidate molecule to each of the plurality of nucleic acid scaffolds, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule and the second candidate molecule of each of the plurality of screening nucleic acid scaffolds are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other; and (e) by repeating (d), determining the force required to achieve an elongation length of the screening nucleic acid scaffold equal to the reference elongation length, wherein the force is at 0.(f) Contacting the plurality of third candidate molecules with the plurality of screening nucleic acid scaffolds; (g) Determining, by repeating (b), the elongation length of each of the plurality of screening nucleic acid scaffolds in response to the same force applied in (e) in the presence of at least one of the plurality of third candidate molecules; and (h) Calculating the difference between the reference elongation length and the elongation length of each of the plurality of screening nucleic acid scaffolds, wherein a non-zero difference indicates that there is a binding interaction between the third candidate molecule and the first candidate molecule and the second candidate molecule under the applied force, and wherein a zero difference indicates that there is no binding interaction between: (A) the third candidate molecule and the first candidate molecule, (B) the third candidate molecule and the second candidate molecule, or (C) the third candidate molecule under the applied force, and the first candidate molecule and the second candidate molecule. In some embodiments, at least two nucleic acid scaffolds in the nucleic acid scaffold contain barcode sequences located at different positions relative to each other. In some embodiments, at least two nucleic acid scaffolds in the nucleic acid scaffold contain barcode sequences that are distinct from each other. In some embodiments, the method further includes determining the identity of consecutive polynucleotide sequences (e.g., hairpin nucleic acids) based on the barcode prior to (b). In some embodiments, the identity of consecutive polynucleotide sequences (e.g., hairpin nucleic acids) is determined by detecting the position of one or more modified nucleotides in the barcode.

[0016] This document also provides a method for determining the binding interaction between a first candidate molecule and a second candidate molecule, the method comprising: (a) providing a nucleic acid scaffold as described herein, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of a chamber of the device; (b) providing a device comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface; and (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; and (d) determining that the nucleic acid scaffold responds to a value less than 0.0 in the absence of the first candidate molecule and the second candidate molecule. (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other; (f) by repeating (d), determining the amplitude of the Brown noise of the screening nucleic acid scaffold in response to the same force used in the absence of the first candidate molecule and the second candidate molecule; and (g) identifying an interaction event between the two molecules, wherein the amplitude of the Brown noise is reduced compared to the reference amplitude under the same force in the absence of the molecule.

[0017] This document also provides a method for determining binding interactions between a first candidate molecule, a second candidate molecule, and a third candidate molecule, the method comprising: (a) providing a nucleic acid scaffold as described herein, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of a chamber of the device; (b) providing a device comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and (ii) a force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; (d) determining a reference amplitude of the nucleic acid scaffold in response to Brownian noise of a force less than 0.01 pN in the absence of the first candidate molecule and the second candidate molecule; (e) (d) Contact the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other; (f) Contact the screening nucleic acid scaffold with a third candidate molecule in solution; (g) Determine the amplitude of the Brown noise of the screening nucleic acid scaffold in the presence of the third candidate molecule in response to the same force applied in (d); and (h) Identify an event of interaction between the first molecule, the second molecule and the third molecule, wherein the amplitude of the Brown noise is reduced compared to the reference amplitude under the same force in the absence of the first molecule, the second molecule and the third molecule.

[0018] This document also provides a kit for determining the binding interaction between a first candidate molecule and a second candidate molecule. In some embodiments, the kit includes: (a) a nucleic acid comprising: (i) a continuous polynucleotide sequence comprising: (I) a first end comprising: (A) a first adaptor for attachment to a bead, and (B) a first molecule-binding sequence; (II) a second end comprising: (A) a second adaptor for attachment of the nucleic acid to a bottom surface of a device, and (B) a second molecule-binding sequence; (III) an intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises: (A) a first lead-forming sequence comprising a barcode, and (B) a second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence and the second lead-forming sequence are complementary to the first lead-forming sequence. The device includes (c) a hybridization of the first and second lead-forming sequences; (ii) a first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and (iii) a second spacer polynucleotide having a polynucleotide sequence complementary to the second lead-forming sequence, wherein when the nucleic acid is attached as a nucleic acid scaffold to the bottom surface of the device, a force of 10 pN to 30 pN is required to unfold the continuous polynucleotide sequence (e.g., a hairpin nucleic acid) along an axis perpendicular to the bottom surface of the device; and (b) a bead containing an anchoring molecule configured to bind a first adaptor to the first end of the continuous polynucleotide sequence of the nucleic acid. In some embodiments, the first and second molecule-binding sequences each independently comprise small hairpin nucleic acids having a size ranging from 5 to 100 bases. In some embodiments, the kit further includes: (a) a first candidate molecule linked to a first gel sequence, wherein the first gel sequence is complementary to the first molecule-binding sequence; and (b) a second candidate molecule linked to a second gel sequence, wherein the second gel sequence is complementary to the second molecule-binding sequence. In some embodiments, the kit further includes: (a) a first gel sequence comprising a first active group configured to be linked to the first candidate molecule, wherein the first gel sequence is complementary to the first molecule-binding sequence; and (b) a second gel sequence comprising a second active group configured to be linked to the second candidate molecule, wherein the second gel sequence is complementary to the second molecule-binding sequence. In some embodiments, the kit further includes at least one of the first candidate molecule and the second candidate molecule. In some embodiments, the kit further includes a third candidate molecule. In some embodiments, the first spacer polynucleotide and the second spacer polynucleotide are linked to each other via a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop. Attached Figure Description

[0019] The novel features of the exemplary embodiments are specifically set forth in the appended claims. A better understanding of the features and advantages will be obtained by referring to the following detailed description of illustrative embodiments in which the principles of the disclosed system and method are utilized, along with the accompanying drawings, in which:

[0020] Figure 1 The components of an exemplary hairpin nucleic acid according to an embodiment of this disclosure are depicted.

[0021] Figure 2 Non-limiting exemplary structures of two polynucleotide sequences for use in preparing hairpin nucleic acids are provided, comprising: (a) a first polynucleotide sequence (SEQ ID NO: 1) (5' to 3') encoding a first adaptor 106, a first molecular binding sequence 104, a first linker sequence 112, and a first lead-forming sequence 108; and (b) a second polynucleotide sequence (SEQ ID NO: 2) (5' to 3') encoding a second lead-forming sequence 109, a second linker sequence 113, a second molecular binding sequence 105, and a second adaptor 107. The first lead-forming sequence 108 and the second lead-forming sequence 109 are complementary to each other, thus forming a double-stranded region upon hybridization. The first lead-forming sequence 108 and the second lead-forming sequence 109 can be linked to each other via a loop 110 to form a hairpin nucleic acid 100. The first linker sequence 112 and the second linker sequence 113 anchor Y-shaped spacer polynucleotides to form a 4-way link (e.g., a holiday link). The two molecular binding sequences are neither identical nor complementary to each other. The 5' end of the first polynucleotide sequence is conjugated with biotin, which can be used to anchor the hairpin nucleic acid to the bead. Similarly, a second adaptor sequence can be used to attach the hairpin nucleic acid to a feature on the bottom surface of the device, wherein the feature is an oligonucleotide complementary to the nucleotide sequence of the second adaptor 107. The letters K, X, Y, and J represent any natural or modified nucleotide (ACGT).

[0022] Figure 3 An exemplary nucleotide sequence engineered to include a barcode is shown. In short, as... Figure 3 As shown, the nucleotide sequence is engineered to contain multiple CCwGG (SEQ ID NO: 3) sequence motifs. When the plasmid containing the nucleotide sequence is propagated in *E. coli*, the DCM methyltransferase present in *E. coli* methylates the second cytosine within the CCwGG (SEQ ID NO: 3) sequence, thereby generating a specific epigenetic barcode that can be decoded (identified) using an anti-5mC antibody on the device described herein. The nucleotide sequence also contains a non-palindromic restriction site BsaI at either end to connect the linking sequence and the loop.

[0023] Figure 4 Twenty exemplary barcodes are shown, which can be generated in a computer simulation by placing constant sites at different locations within a 200-base fragment. The exemplary constant sites contain the nucleotide sequence CCwGG (SEQ ID NO: 3). As shown, two constant sites spaced by 10 and 18 nucleotides, respectively, were incorporated into all barcodes. The nucleotide sequences were methylated by a DCM methyltransferase present in *E. coli* and detected using an antibody against 5-mC. To align different nucleotides with predicted sequences, two constant sites present in all nucleotides were used. The number of CCwGG (SEQ ID NO: 3) sequences within each nucleotide and the distance between the two sites can be varied, allowing the generation of a library of nucleotide fragments for multiplexing.

[0024] Figure 5 The components of an exemplary spacer polynucleotide according to an embodiment of the present disclosure are described.

[0025] Figure 6 Non-limiting exemplary structures of two spacer polynucleotide chains for use in preparing Y-shaped spacer polynucleotides are depicted, wherein the two spacer polynucleotide chains comprise: (a) a first spacer polynucleotide chain (SEQ ID NO: 5) encoding a polynucleotide sequence (5' to 3') complementary to consecutive portions of a first linker sequence binding region (505) and a first lead-forming sequence binding region (503); and (b) a second spacer polynucleotide chain (SEQ ID NO: 6) encoding a polynucleotide sequence (5' to 3') complementary to consecutive portions of a second lead-forming sequence binding region (504) and a second linker sequence binding region (506). The two polynucleotides can be annealed to form a Y-shape, which can be linked to the same barcode sequence used to construct a hairpin sequence. The first linker sequence and the second linker sequence anchor the first spacer polynucleotide chain and the second spacer polynucleotide chain, respectively, to form a 4-way link (e.g., a holiday link). The letters X' and Y' represent any natural or modified nucleotide (ACGT).

[0026] Figure 7A non-limiting exemplary system comprising a holiday link formed between a hairpin nucleic acid and a spacer polynucleotide is illustrated. As shown, the hairpin nucleic acid is attached to a bead (SEQ ID NO: 11) and a feature portion of the device surface. When sufficient force is applied to the bead, the holiday link is disengaged, resulting in chain invasion. After invasion, a first lead-forming sequence and a second lead-forming sequence of the hairpin nucleic acid hybridize with the first and second spacer polynucleotide chains, respectively. Arrows indicate the orientation of each component of the exemplary system and the protruding ends required for cloning barcode sequences (SEQ ID NO: 4 as an example of digestion with the restriction enzyme BsaI).

[0027] Figure 8 A to Figure 8 D illustrates a graphical representation of the method for identifying hairpin nucleic acid 100 using the system described herein. (See diagram below.) Figure 8 As shown in Figure A, under low strength (below 12 pN), the hairpin sequence hybridizes completely, and the antibody cannot bind methylated cytosine. Figure 8 As shown in Figure B, the hairpin molecule unfolds when the force is increased (above 20 pN). Therefore, the methylated cytosine (5 mC) is now exposed, and the anti-5 mC antibody approaches these modified bases to bind to them. The force is then reduced to 12 pN or below, resulting in the reformation of the hairpin. Thus, as... Figure 8 As shown in Figure C, the fork is momentarily blocked when the antibody binds. Record the location of this blockage. Figure 8 As shown in D, the first antibody conjugate is then removed, and the fork continues its path toward the next antibody conjugate, recording the location of subsequent blockages. Since this process is non-destructive, this cycle can be repeated multiple times until all locations have been extracted.

[0028] Figure 9 The diagram shows alignments over 100 cycles of hairpin opening and closing in the presence of the antibody. Each star and dashed line represents a location where methylated cytosine has been detected. These locations can then be aligned with predicted sequences to identify the barcode sequence.

[0029] Figure 10The results of identifying nucleotides by decoding barcodes are shown. Seven different plasmids, each containing a different barcode sequence, were mixed in equimolar amounts and digested with BsaI to produce a mixture of seven different nucleotide fragments. Y-shaped nucleic acids and loops were then ligated at either end of the fragments, and the resulting hairpin nucleic acids were attached to paramagnetic beads. The paramagnetic beads were injected into a flow cell and allowed to bind to the surface. After removing unbound beads, an antibody against 5-mC was injected into the flow cell, and force cycling was performed to detect the positions of all methylated cytosines on all nucleotides. After more than one hundred cycles, the choke sites were extracted and mapped to their expected sequences by mapping the experimental chokes to their expected positions. HP = hairpin

[0030] Figure 11 A to Figure 11 C shows a graphical representation of a method for preparing a nucleic acid scaffold by performing chain intrusion from hairpin nucleic acid 100 located between the bead and a feature portion on the surface of the device.

[0031] Figure 12 The results of successful invasion are shown. The force was set constant at 5 pN when the hairpin nucleic acid and the corresponding invasion spacer polynucleotide were present in the flow cell. Starting from the arrow, the characteristic portion of the signal changes, corresponding to the invasion of the middle portion 103 of the hairpin nucleic acid 100 by the spacer polynucleotide, indicating successful strand invasion.

[0032] Figure 13 A to Figure 13 C illustrates a graphical representation of the method for determining the binding interaction between two candidate molecules using the system described herein.

[0033] Figure 14 Experimental traces of the interaction between two proteins (KU70 / 80 and APFL) on a double-stranded substrate present on the scaffold DNA are shown. Four different nucleotide sequences (SEQ ID NO: 15-18) were used as the double-stranded substrate. The interaction between the two proteins resulted in a shortening of the Z-shift of the bead, as shown in the box. Once the interaction was disrupted, the scaffold stretched completely and reached a length similar to that observed in the control experiment (in buffer only, without the proteins).

[0034] Figures 15A to 15B An exemplary trace used to determine the binding dynamics between two candidate molecules is shown. The force is maintained at 0.1 pN, which allows the scaffold to be flexible enough for the two proteins to interact (if they can interact). Under low force, the beads attached to the scaffold move due to Brownian motion, the amplitude of which roughly corresponds to the length of the scaffold. During interaction, the distance between the beads and the surface decreases, which restricts Brownian motion. Therefore, the binding dynamics related to the duration of the interaction are determined by tracking the movement of the beads over time.

[0035] Figure 16 Exemplary variations in the Brownian motion of beads anchored to a nucleic acid scaffold are shown in the presence and absence of forces relative to the device. Both the human receptor ACE2 and SARS-CoV2 RBD domains are labeled with oligonucleotides that allow them to be anchored to molecular binding sequences 104 and 105, respectively. The proteins exhibit the ability to interact when the force is reduced to <0.1 pN. A characteristic feature of the interaction is the reduction in the amplitude of the Brownian noise (by...). Figure 16 (Indicated by the arrow in the diagram). The interaction is disrupted as the force increases (approximately 2 pN). The interaction is observed again when the force is reduced to a low level (<0.1 pN), because this is a non-destructive process (by...). Figure 16 (The arrow in the text indicates this).

[0036] Figure 17 A generic barcode sequence is shown that can serve as the basis for all subsequent barcodes. This sequence can also be used to generate generic chain-intrusion hairpins. In short, as... Figure 17 As shown, the nucleotide sequence (SEQ ID NO: 35) was engineered to contain two CCwGG (SEQ ID NO: 3) sequence motifs separated by 20 and 10 bases at the 5' and 3' ends, respectively, which will always be included in all barcodes and therefore always methylated in all generated barcode fragments. Then, fourteen CawGG (SEQ ID NO: 54) sites were inserted between these reference positions at varying intervals between each site. A sequence with a consistent GC content throughout the fragment (total GC content of 43%) was also produced. When new barcodes are needed, any of these positions (between positions 1 and 14) can be mutated, thus giving a theoretically maximum number of barcodes up to 2... 14 A fragment (SEQ ID NO: 3) can be converted to CCwGG, which is methylated by *E. coli* DCM methyltransferase when a plasmid containing this nucleotide sequence is propagated in bacteria. When the fragment is isolated from the plasmid, it generates a specific epigenetic barcode that can be decoded on a platform using an anti-5mC antibody. The nucleotide sequence also contains a non-palindromic restriction site BsaI at either end to generate a 4-base overhang required for the linker sequence and the loop.

[0037] Figure 18 This demonstrates how four or five CawGG sequences can be altered to CCwGG (SEQ ID NO: 39-44) to obtain... Figure 3The sequences shown generate five different methylation patterns and an example of a universal chain-invading fragment. As shown, two constant sites, spaced by 10 and 20 nucleotides respectively, are transplanted into all barcodes as references at the 3' and 5' ends. The nucleotide sequences are methylated by DCM methyltransferase present in *E. coli* and detected using an antibody against 5-mC. To align different nucleotides with their corresponding predicted sequences, four constant sites (two at each end) present in all barcodes are used. The number of CCwGG (SEQ ID NO: 3) sequences within each fragment can vary, allowing the generation of libraries of nucleotide fragments for multiplexing, with a maximum number of sequences of 2. 14 .

[0038] Figure 19A A graphical representation of the components of a nucleic acid scaffold from a hairpin nucleic acid 100 located between a bead and a feature portion on the surface of the device is provided, wherein the first molecule binding sequence 104 and the second molecule binding sequence 105 are each a small hairpin nucleic acid. Figure 19B A graphical representation of hairpin nucleic acids is shown, which allows for ensuring protein / molecule anchoring to a scaffold. (See diagram.) Figure 19B As shown, the hairpin nucleic acid 104 comprises a first molecular binding gel complementary strand 601 and a second molecular binding gel complementary strand 602 connected to each other via molecular binding complementary loops 605. The first molecular binding gel complementary strand 601 includes a first molecular binding lead-forming sequence binding region 603. The second molecular binding gel complementary strand 602 includes a second molecular binding lead-forming sequence binding region 604. The GC content of the lead sequence 602 and its complementary sequence 603 is greater than 90% to ensure that opening this sequence does not interfere with opening the barcode sequence. The structure of sequence 105 is similar to... Figure 19B The structure presented will differ from sequence 104, except that sequences 601 and 602 will be different to allow anchoring to specific target molecules. Figure 19C An exemplary gel polynucleotide covalently linked to a candidate molecule is shown. Figure 19C As shown, the gel polynucleotide 700 comprises a first gel spacer polynucleotide chain 701 and a second gel spacer polynucleotide chain 702 connected to each other via gel rings 707. The first gel spacer polynucleotide chain 701 includes a first gel linker sequence binding region 705 and a first gel lead-forming sequence binding region 703. The second gel spacer polynucleotide chain 702 includes a second gel linker sequence binding region 706, a second gel lead-forming sequence binding region 704, and a single-stranded sequence 708 between the hairpin and the candidate molecule. The second gel linker sequence binding region 706 can be covalently attached to the candidate molecule.

[0039] Figure 20A The expected binding traces of the gel sequence linked to the candidate molecule are shown. Figure 20B It shows Figure 1The prototype sequences 104 and 105 are presented in the image. These sequences consist of two variable sequences (parts 1 and 3) of 12 bases on either side of the hairpin sequence (part 2) to allow specific proteins to anchor to specific scaffold structures. Figure 20C The prototype sequences of the oligonucleotides covalently attached to the target molecule are shown. Sequences 3 and 5 correspond to the complementary sequences present on the scaffold structure. Figure 20B The sequences (parts 1 and 3) will allow specific target molecules to be anchored to different structures. Sequence 1 (with the associated TTT of sequence 2) can be located at the 5' or 3' end of the invading oligonucleotide. The sequences and their length can be between 10 and 50 nucleotides to allow for system flexibility. Figure 20D An example of how to monitor the anchoring of a target molecule is shown. Two small closed jumps (indicated by arrows) at approximately 30 nm are observed, as well as a closed jump caused by the opening of a hairpin. After the addition of a first invading oligonucleotide (for this proof-of-concept, no target molecule is attached to the oligonucleotide), one of the two jumps disappears, and only one jump is observed. Upon the addition of a second oligonucleotide, the second closed jump also disappears due to the invading of a second small hairpin. The letters X and Y represent any natural or modified nucleotide (ACGT).

[0040] Figure 21 The results of identifying different segments by decoding a barcode are shown. Figure 18 The six fragments described were sequenced, cloned into plasmids, and propagated in *E. coli*, such that all CCwGG sites within these sequences were methylated. After parallel digestion of the plasmids (each containing a different barcode sequence) with BsaI, Y-shaped nucleic acids and loops were ligated to both ends of these fragments. Six resulting nucleic acid hairpins were attached to paramagnetic beads and mixed in an equimolar ratio. The paramagnetic beads were injected into a flow cell and allowed to bind to the surface. After removing unbound beads, an antibody against 5-mC was injected into the flow cell, and force cycling was performed to detect the location of all methylated cytosine on all hairpins. After more than one hundred cycles, the choke sites were extracted and mapped to their expected sequences by mapping the experimental chokes to their expected locations. BC = barcode

[0041] Figure 22 A to Figure 22 C provides a graphical representation of a method for preparing a nucleic acid scaffold by performing chain intrusion from a hairpin nucleic acid 100 located between a bead and a feature portion on the surface of the device. In short, a universal chain-intrusion hairpin (SEQ ID. NO: 35) is injected into a flow cell, and the hairpin binds through complementarity between a first linker sequence and a second linker sequence.

[0042] Figure 23 It shows Figure 22 A to Figure 22An exemplary trajectory of an event described in C. In short, as... Figure 23 As shown, when the force is increased between 5pN and 10pN, the formed holiday connections are eliminated, and the intrusion can be observed through a molecular extension of about 200 nm.

[0043] Figure 24 Chain invasion efficiency in hairpin nucleic acids containing universal barcode sequences is shown. In short, five hairpin nucleic acids (SEQ ID NO: 40-44) were generated and chain invasion efficiency was analyzed.

[0044] Figure 25A A graphical representation of how the system described herein can be used to probe molecular interactions to determine the binding interaction between two candidate molecules is shown. In short, after both target molecules have attached to the scaffold structure, the force is reduced to 0.01 pN to allow the two molecules to interact. The force is then increased to a value between 0.5 pN and 20 pN, depending on the strength of the interaction. If the two molecules interact before this increase in force, full extension of the dsDNA is prevented, and an intermediate position will be observed. After the interaction is disrupted, the dsDNA scaffold will extend fully, causing a sudden jump in the bead position. The energy (entropy, enthalpy, and ΔG) of the interaction can then be extracted using the time and force required to disrupt this interaction. Figure 25B The traces of the formation of the complex consisting of KU70 / 80 (which binds at the double-strand break) and the protein APFL are shown. In short, two adaptors (each consisting of two oligonucleotides that independently form a double-stranded region and a single-stranded gel sequence complementary to the first and second adaptor sequences located on either side of the scaffold) were injected into a flow cell, and the position of the beads was recorded over 100 force cycles (only 14 cycles are shown in this figure). In the absence of any protein, the scaffold fully elongates when the force is increased to 2 pN (top inset). After injection of the Ku70 / 80 complex and the protein APFL, the Ku70 / 80 complex forms at the blunt ends of both adaptors, and the protein APFL bridges these complexes. This can be observed by the transiently shorter elongation of the scaffold when the complex forms (indicated by the box, bottom inset).

[0045] Figure 26AA graphical representation of how the binding kinetics between two candidate molecules can be determined under a constant low force is shown. The force is kept at 0.01 pN, which allows the scaffold to be flexible enough for the two proteins to interact (if they can interact). Under this low force, the beads attached to the scaffold move due to Brownian motion, the amplitude of which corresponds to the length of the scaffold (0.3 nm per base pair, and the scaffold consists of 900 bp or 250 nm displacements). During interaction, the distance between the beads and the surface decreases, which restricts Brownian motion (the scaffold decreases to only 300 bpm, which represents only 100 nm displacement). The binding kinetics can be determined, corresponding to how many events (Kon) are observed during the duration of the non-interacting state, and Koff corresponds to the average of all detected interactions. Figure 26B The true trace of the interaction between the human receptor protein ACE2 and the wild-type SARS-CoV-2 virus receptor-binding protein (RBD) is shown. Five interactions between these two proteins detected within this 5-minute recording are indicated by red lines. Figure 26C The diagram shows two 5-minute phases separated by a phase at 20 pN (to reset all molecules to a non-interacting state). Because the process is non-destructive, interactions can be observed after this "reset" phase.

[0046] Figure 27 An example of a tertiary complex formation is shown between the E3 ligase CERBLON (complexed with its binding partner DDB1), the novel target GSPT1, and the molecular glue CC885. Short interaction events (not visible in this figure) can be observed in the absence of the molecular glue, but the addition of the molecular glue greatly stabilizes the complex. In fact, the complex is only disrupted when the force increases to 20 pN, and it reforms within seconds when the force decreases to 0.01 pN.

[0047] Figure 28 An example of a tertiary complex formation is shown between the E3 ligase CERBLON (complexed with its binding partner DDB1), the novel target GSPT1, and the molecular glue thalidomide. The formation of the tertiary complex can be observed in the presence of the molecular glue. In contrast to the molecular glue CC885, the interaction is weaker and can be disrupted even under low forces.

[0048] Figure 29The formation of a tertiary complex is shown between the E3 ligase CERBLON (complexed with its binding partner DDB1), the novel substrate IKAROS1, and the molecular compound pomalidomide. In the absence of the small molecule, there is no interaction or the interaction is very short (indicated by small triangles). Upon the addition of the molecular compound pomalidomide, a strong tertiary complex is formed (indicated by arrows). This complex is so strong that it can withstand forces up to 2 pN (indicated by asterisks).

[0049] Figure 30 This paper demonstrates the multiplexing capability of the system described herein. Two distinct barcodes were constructed, each with the same complementary sequence to anchor the ACE2 protein and two distinct single-stranded sequences at the other end: one for the wild-type RBD protein and a second sequence specific to the RBDΔ mutant sequence. Once randomly attached to a flow cell, the barcodes were “read” to determine the coordinates of each barcode within the flow cell. A universal strand intrusive hairpin was then injected into the flow cell, and the force was set to 5 pN to allow the hairpin to transform into a dsDNA scaffold. ACE2 and the wild-type RBD were injected into the flow cell first, followed by constant-force cycling. Interactions were observed only on molecules specific to the wild-type RBD. No interactions were observed on scaffolds specific to the RBDΔ mutant. The RBDΔ mutant was then injected into the flow cell, and low-force cycling was repeated. Interactions on both scaffolds (wild-type RBD and mutant RBD) were observed simultaneously in the same flow cell, demonstrating that the binding kinetics of different interactions can be measured simultaneously within the same flow cell. Detailed Implementation

[0050] Overview

[0051] This disclosure relates to the analysis of molecular interactions between two or more molecules in a highly parallel manner at the single-molecule level using magnetic tweezers. There is a great need to understand the interactions between different proteins or between proteins and small molecules, but these interactions often rely on indirect methods such as immunoprecipitation or co-localization.

[0052] This document discloses a method for determining binding interactions between two or more candidate molecules in real time and at the single-molecule level. In some embodiments, the method involves anchoring the candidate molecule (protein or nucleic acid) to a bead. In some embodiments, the method involves generating a force sufficient to pull and stretch the candidate molecule. Depending on the structure of the candidate molecule, different signals are expected. For example, in the case of a hairpin, a force reaching >20 pN allows for mechanical denaturation of the nucleic acid and the formation of two distinct and complementary single strands. Therefore, in some embodiments, the method is used to analyze the physical properties of candidate molecules (e.g., DNA, RNA, or combinations thereof). Alternatively, in some embodiments, the method is used to determine the location of interactions between various enzymes (e.g., polymerases, helicases, topoisomerases) and candidate molecules. Furthermore, in some embodiments, the method is used to analyze the binding kinetics of one candidate molecule (e.g., oligonucleotides or proteins (e.g., antibodies)) to another candidate molecule. However, most molecular interactions between proteins or small molecules are relatively weak (≤5 pN). Such molecular interactions are difficult to analyze on magnetic tweezers (poor resolution at low forces due to high Brownian motion). Specifically, weak interactions can result in excessive noise, preventing the extraction of a suitable signal (signal-to-noise ratio S / N is too low). The methods and systems disclosed herein advantageously enable: analysis of molecular interactions at the single-molecule level; parallel measurement of multiple interactions; determination of interaction locations by guiding the positions of candidate molecules (proteins or small molecules) in a solution coupled to the system; and amplification of signals (interacting or non-interacting) to well above the noise level to identify molecular interactions where the interaction force is ≤0.1 pN.

[0053] system

[0054] This document provides a system comprising a nucleic acid scaffold. In some embodiments, the nucleic acid scaffold comprises a hairpin nucleic acid. In some embodiments, the hairpin nucleic acid comprises two molecular binding sequences and two spacer region binding sequences. In some embodiments, the two spacer region binding sequences are looped together to form a continuous polynucleotide sequence. In some embodiments, each of the two spacer region binding sequences comprises a lead-forming sequence and an optional linker sequence. In some embodiments, the nucleic acid scaffold also comprises one or two spacer region polynucleotides. In some embodiments, the nucleic acid scaffold also comprises two candidate molecules, each linked to a candidate molecule. In some embodiments, the nucleic acid scaffold is anchored to a bead. In some embodiments, the nucleic acid scaffold is anchored to the bead via non-covalent interactions, wherein a first end comprises a first adaptor, wherein the bead comprises a first handle sequence conjugated to the bead, wherein the first adaptor and the first handle sequence are complementary to each other. In some embodiments, the system further includes a device (e.g., a chamber) comprising one or more feature portions. In some embodiments, the feature portions of the device described herein are configured to anchor the nucleic acid scaffold described herein. In some embodiments, the nucleic acid scaffold is attached to a feature portion of the device via non-covalent interactions, wherein the second end includes a second adapter, and the feature portion includes a second handle sequence that engages with the feature portion, wherein the second adapter and the second handle sequence are complementary to each other. In some embodiments, the device is capable of applying force to a bead, thereby causing the bead to move. In some embodiments, the feature portion is operatively connected to a sensor capable of measuring the movement of the bead. In some embodiments, the movement of the bead is vertical.

[0055] hairpin nucleic acid

[0056] This document discloses hairpin nucleic acids capable of being positioned between a bead and a feature portion of a device surface. In some embodiments, the hairpin nucleic acid is a continuous polynucleotide sequence. In some embodiments, the hairpin nucleic acid has a Y-shape to allow anchoring to the bead on one side and to the surface at the other end. In some embodiments, the hairpin nucleic acid comprises two molecular binding sequences, two spacer binding sequences, and two adaptors. In some embodiments, the spacer binding sequences comprise two lead-forming sequences, a loop, and two linker sequences. In some embodiments, the lead-forming sequences comprise one or more barcodes. In some embodiments, the hairpin nucleic acid comprises a polynucleotide with a size between 10 and 1500 bases. In some embodiments, the hairpin nucleic acid comprises a polynucleotide with a size between 15 and 2000 bases. In some embodiments, the hairpin nucleic acid comprises a polynucleotide with a size between 50 and 1000 bases.

[0057] In some embodiments, the hairpin nucleic acid molecule is chemically synthesized. In some embodiments, the hairpin nucleic acid molecule is produced via in vitro transcription. In some embodiments, the hairpin nucleic acid molecule is produced via polymerase chain reaction. In some embodiments, the hairpin nucleic acid molecule is purified from cells. In some embodiments, the hairpin nucleic acid molecule is chemically synthesized, produced via in vivo transcription, produced via polymerase chain reaction, purified from cells, or a combination thereof.

[0058] Figure 1An exemplary structure of a hairpin nucleic acid 100 is shown. As shown, the hairpin nucleic acid 100 comprises a continuous polynucleotide sequence including a first end 101, a second end 102, and an intermediate portion 103 between the first end 101 and the second end 102. As shown, the first end 101 includes a first molecule-binding sequence 104, and the second end 102 includes a second molecule-binding sequence 105. In some embodiments, the first molecule-binding sequence 104 hybridizes to a first gel sequence of a first candidate molecule. In some embodiments, the second molecule-binding sequence 105 hybridizes to a second gel sequence of a second candidate molecule. In some embodiments, the first end 101 is anchored to a bead. In some embodiments, the first end 101 is anchored to the bead via a covalent interaction or a non-covalent interaction (e.g., streptavidin-biotin interaction). Alternatively, in some embodiments, the first end 101 includes a first adaptor 106 located at the end of the first end 101. In some embodiments, the first adaptor 106 is capable of hybridizing with a first handle sequence conjugated to the bead, wherein the first adaptor 106 and the first handle sequence are complementary to each other. In some embodiments, the second end 102 is attached to a feature portion on the surface of the device. In some embodiments, the second end 102 is attached to the feature portion by covalent or non-covalent interactions (e.g., strep tag-biotin interactions). Alternatively, in some embodiments, the second end 102 includes a second adaptor 107 located at the end of the second end 102. In some embodiments, the second adaptor 107 is capable of hybridizing with a second handle sequence conjugated to the feature portion, wherein the second adaptor 107 and the second handle sequence are complementary to each other. As shown, the intermediate portion 103 is located between a first molecule-binding sequence 104 and a second molecule-binding sequence 105. In some embodiments, the intermediate portion 103 includes a hairpin structure. In some embodiments, the intermediate portion 103 includes a continuous nucleotide sequence comprising a first spacer region binding sequence (not shown) and a second spacer region binding sequence (not shown), wherein the first spacer region binding sequence is located between the first molecule-binding sequence 104 and the second spacer region binding sequence. Therefore, in some embodiments, the second spacer binding sequence is located between the first spacer binding sequence and the second molecule binding sequence 105. In some embodiments, the first spacer binding sequence comprises a first lead-forming sequence 108, and the second spacer binding sequence comprises a second lead-forming sequence 109. Therefore, in some embodiments, the intermediate portion comprises a continuous nucleotide sequence comprising the first lead-forming sequence 108 and the second lead-forming sequence 109. In some embodiments, the nucleotide sequences of the first lead-forming sequence 108 and the second lead-forming sequence 109 are complementary to each other.In some embodiments, the first spacer binding sequence and the second spacer binding sequence are connected by a loop 110 to form a continuous polynucleotide sequence. Therefore, in some embodiments, the loop 110 is located between the first lead-forming sequence 108 and the second lead-forming sequence 109. In some embodiments, the first lead-forming sequence 108, the loop 110, and the second lead-forming sequence 109 form a hairpin structure. For example... Figure 1 As shown, in some embodiments, the first spacer region binding sequence further includes a first linker sequence 112 located between the first molecule binding sequence 104 and the first lead-forming sequence 108. Similarly, as Figure 1 As shown, in some embodiments, the second spacer region binding sequence further includes a second linker sequence 113 located between the second molecular binding sequence 105 and the second lead-forming sequence 109. The first linker sequence 112 and the second linker sequence 113 may not be complementary to each other. Figure 20B It shows Figure 1 The prototype sequences of sequences 104 and 105 are presented in the image.

[0059] Figure 2 Non-limiting exemplary structures of two polynucleotide sequences for use in preparing Y-shaped nucleic acids, which can be further used to prepare hairpin nucleic acids, are provided. These two polynucleotide sequences comprise: (a) a first polynucleotide sequence (SEQ ID NO: 1) (5' to 3') encoding a first adaptor (106), a first molecule-binding sequence (104), a first linker sequence (112), and a first lead-forming sequence (108); and (b) a second polynucleotide sequence (SEQ ID NO: 2) (5' to 3') encoding a second lead-forming sequence (109), a second linker sequence (113), a second molecule-binding sequence (105), and a second adaptor (107). Exemplary nucleotide sequences (5' to 3') encoding the second lead-forming sequence (109), the second linker sequence (113), the second molecule-binding sequence (105), and the second adaptor (107) are provided in SEQ ID NO: 33.

[0060] Molecular binding sequence

[0061] In some embodiments, the first end 101 and the second end 102 of the hairpin nucleic acid 100 respectively comprise a first molecular binding sequence 104 and a second molecular binding sequence 105. In some embodiments, the first molecular binding sequence 104 and the second molecular binding sequence 105 are not identical to each other. In some embodiments, the first molecular binding sequence 104 and the second molecular binding sequence 105 are not complementary to each other. In some embodiments, the first molecular binding sequence 104 and the second molecular binding sequence 105 comprise the middle portion 103 of the hairpin nucleic acid between the first molecular binding sequence 104 and the second molecular binding sequence 105. In some embodiments, the first molecule binding sequence 104 and the second molecule binding sequence 105 independently have 5 to 100 bases, 5 to 80 bases, 5 to 60 bases, 5 to 40 bases, 5 to 20 bases, 10 to 100 bases, 10 to 80 bases, 10 to 60 bases, and 10 to 40 bases. Sizes ranging from 10 to 20 bases, 20 to 100 bases, 20 to 80 bases, 20 to 60 bases, 20 to 40 bases, 40 to 100 bases, 40 to 80 bases, 40 to 60 bases, 60 to 100 bases, 60 to 80 bases, or 80 to 100 bases.

[0062] In some embodiments, the molecular binding sequence comprises a small hairpin nucleic acid. In some embodiments, the small hairpin nucleic acid has the following lengths: 5 to 100 bases, 5 to 80 bases, 5 to 60 bases, 5 to 40 bases, 5 to 20 bases, 10 to 100 bases, 10 to 80 bases, 10 to 60 bases, 10 to 40 bases, 10 to 20 bases. Sizes ranging from 20 to 100 bases, 20 to 80 bases, 20 to 60 bases, 20 to 40 bases, 40 to 100 bases, 40 to 80 bases, 40 to 60 bases, 60 to 100 bases, 60 to 80 bases, or 80 to 100 bases. An exemplary hairpin nucleic acid containing a small hairpin nucleic acid is shown in... Figure 19A middle. Figure 19B A graphical representation of the molecular binding sequence comprising a hairpin nucleic acid is shown. In some embodiments, the hairpin nucleic acid allows for ensuring protein / molecule anchoring to the nucleic acid scaffold described herein.

[0063] Interval region binding sequence

[0064] In some embodiments, the middle portion 103 of the hairpin nucleic acid 100 includes two spacer binding sequences. In some embodiments, the two spacer binding sequences form a continuous polynucleotide sequence. In some embodiments, the two spacer binding sequences are located between two molecular binding sequences of the hairpin nucleic acid 100. In some embodiments, the two spacer binding sequences include a first spacer binding sequence and a second spacer binding sequence. In some embodiments, at least a portion of the first spacer binding sequence is complementary to at least a portion of the second spacer binding sequence. In some embodiments, the first and second spacer binding sequences form a continuous polynucleotide sequence when joined together, wherein the first and second spacer binding sequences can form a hairpin structure when they hybridize with each other. In other words, in some embodiments, the first and second spacer binding sequences allow the formation of a continuous polynucleotide sequence when they hybridize with each other and are joined with lead sequences 108 and 109 and loop sequence 110, wherein the first and second spacer binding sequences form a hairpin structure. In some embodiments, each of the two spacer binding sequences includes a lead-forming sequence. Therefore, in some embodiments, two lead-forming sequences form a hairpin structure when connected to each other by a loop. In some embodiments, the lead-forming sequences contain one or more barcodes.

[0065] Lead formation sequence

[0066] In some embodiments, the first spacer region binding sequence and the second spacer region binding sequence of the hairpin nucleic acid 100 respectively comprise a first lead-forming sequence 108 and a second lead-forming sequence 109. Therefore, in some embodiments, the first lead-forming sequence 108 and the second lead-forming sequence 109 are located between the two molecular binding sequences of the hairpin nucleic acid 100. In some embodiments, the first lead-forming sequence 108 and the second lead-forming sequence 109 are complementary to each other. In some embodiments, the first lead-forming sequence 108 and the second lead-forming sequence 109 hybridize to each other. In some embodiments, the size of each of the two lead-forming sequences is from 5 to 500 bases, 5 to 400 bases, 5 to 300 bases, 5 to 200 bases, 5 to 100 bases, 5 to 80 bases, 5 to 60 bases, 5 to 40 bases, 5 to 20 bases, and 10 to 500 bases. 10 bases to 400 bases, 10 bases to 300 bases, 10 bases to 200 bases, 10 bases to 100 bases, 10 bases to 80 bases, 10 bases to 60 bases, 10 bases to 40 bases, 10 bases to 20 bases, 20 bases to 500 bases, 20 bases to 400 bases, 20 bases to 300 bases, 20 bases Up to 200 bases, 20 to 100 bases, 20 to 80 bases, 20 to 60 bases, 20 to 40 bases, 40 to 500 bases, 40 to 400 bases, 40 to 300 bases, 40 to 200 bases, 40 to 100 bases, 40 to 80 bases, 40 to 60 bases The range of 60 to 500 bases, 60 to 400 bases, 60 to 300 bases, 60 to 200 bases, 60 to 100 bases, 60 to 80 bases, 80 to 500 bases, 80 to 400 bases, 80 to 300 bases, 80 to 200 bases, or 80 to 100 bases.

[0067] barcode

[0068] In some embodiments, the lead-forming sequence described herein contains one or more barcodes. In some embodiments, decoding of one or more barcodes reveals an underlying nucleotide sequence. For example, in some embodiments, an epigenetic modification pattern of the hairpin nucleic acid is used to decode the barcode. In some embodiments, an epigenetic modification pattern is generated using a protein (e.g., a monoclonal antibody) that binds to the epigenetically modified nucleotide. Non-limiting examples of epigenetic modifications of DNA include 3-methylcytosine (3mC) modification, 4-methylcytosine (4mC) modification, 5-methylcytosine (5mC) modification, 5-hydroxymethylcytosine (5hmC) modification, 5-formylcytosine (5fC) modification, 5-carboxycytosine (5caC) modification, 6-methyladenosine (m6A) modification, or combinations thereof. Non-limiting examples of epigenetic modifications of RNA include 5-hydroxymethyluracil (5hmU) modification, pseudouridine modification, or combinations thereof. Non-limiting examples of epigenetic modifications of DNA or RNA include 3-methylcytosine (3mC) modification, N6-methyladenosine (m6A) modification, or combinations thereof. In some embodiments, the epigenetic modification of DNA includes 5-methylcytosine modification. Thus, in some embodiments, the epigenetically modified nucleotide comprises one or more methylated cytosines. In other embodiments, an enzyme that introduces the epigenetic modification pattern is used. In some embodiments, the epigenetically modified nucleotide comprises one or more methylated adenosines.

[0069] In some embodiments, the barcode includes one or more constant sites. In some embodiments, the barcode includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or more constant sites. In some embodiments, the barcode includes one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more constant sites. In some embodiments, the barcode includes one to fourteen, three to fourteen, seven to fourteen, ten to fourteen, two to twelve, five to twelve, seven to twelve, ten to twelve, one to ten, three to ten, seven to ten, three to eight, five to eight, or two to seven constant sites. In some embodiments, the barcode includes two constant sites. In some embodiments, one or more constant sites are independently present within the first 8 to 20 nucleotides from the 5' end of the lead-forming sequence, within the first 8 to 20 nucleotides from the 3' end of the lead-forming sequence, or both. In some embodiments, at least two constant sites are identical across all barcode fragments. In some embodiments, at least two constant sites are distinct from each other. In some embodiments, at least two constant sites are spaced 5 to 50, 10 to 50, 20 to 50, 30 to 50, 40 to 50, 5 to 40, 10 to 40, 20 to 40, 30 to 40, 5 to 30, 10 to 30, 20 to 30, 5 to 20, 10 to 20, or 5 to 10 bases apart. In some embodiments, the barcode contains two constant sites. In some embodiments, one or more constant sites comprise nucleotide sequences that have undergone epigenetic modification. In some embodiments, one or more constant sites comprise one or more cytosines, one or more adenines, or combinations thereof. In some embodiments, one or more cytosines are methylated by a DCM methyltransferase of *E. coli*. In some embodiments, one or more adenosines are methylated by a DAM methyltransferase of *E. coli*. In some embodiments, one or more nucleotides of SEQ ID NO: 3 in the barcode sequence are methylated by a methyltransferase. In some embodiments, all second Cs of CCwGG (SEQ ID NO: 3) are methylated. In some embodiments, at least the second C of CCwGG (SEQ ID NO: 3) from the 5' end is methylated. In some embodiments, the second C starting from the 5' end of CCwGG (SEQ ID NO: 3) is methylated.

[0070] In some embodiments, the constant site comprises the nucleotide sequence CCwGG (SEQ ID NO: 3). In some embodiments, when a plasmid encoding the constant site having the nucleotide sequence CCwGG (SEQ ID NO: 3) proliferates in E. coli, a DCM methyltransferase present in E. coli methylates the second cytosine within the CCwGG (SEQ ID NO: 3) sequence.

[0071] Figure 3 An exemplary nucleotide sequence of the first hairpin nucleic acid precursor is shown. Figure 4 Twenty exemplary barcodes are shown, which can be generated in a computer simulation by placing two constant site sequences at constant positions at either end of a fragment within a nucleotide, plus a central region containing a varying number of CCwGG sequences (SEQ ID NO: 3) spaced by varying numbers of bases. Therefore, in some embodiments, exemplary barcodes can be generated in a computer simulation by placing two constant site sequences at different positions within a nucleotide. In some embodiments, the constant site comprises a nucleotide sequence of CCwGG (SEQ ID NO: 3). In some embodiments, the constant site comprises a methylated nucleotide sequence of CCwGG (SEQ ID NO: 3).

[0072] In some embodiments, the barcode is generated from a universal barcode sequence, wherein the universal barcode sequence can be used to generate multiple barcodes. In some embodiments, the universal barcode sequence includes two reference sites, each located at both ends of the lead-forming sequence. In some embodiments, the reference sites are nucleotide sequences of CCwGG (SEQ ID NO: 3). In some embodiments, the universal barcode sequence includes one or more constant sites between the reference sites located at both ends of the lead-forming sequence. In some embodiments, the universal barcode sequence includes fourteen constant sites between the reference sites located at both ends of the lead-forming sequence. In some embodiments, the constant sites between the two reference sites of the universal barcode sequence are nucleotide sequences selected from CCwGG (SEQ ID NO: 3) and CawGG (SEQ ID NO: 54). In some embodiments, the constant sites of the CawGG (SEQ ID NO: 54) nucleotide sequence are not methylated when multiplying in bacteria, but can be easily methylated by changing to the nucleotide sequence of CCwGG (SEQ ID NO: 3). In some embodiments, the universal barcode sequence comprises fourteen constant sites located between reference sites at both ends of the lead-forming sequence. Therefore, in such embodiments, the universal barcode sequence contains a total of 14 potential methylation sites, which can be randomly spaced to avoid repetition. Thus, in such embodiments, a barcode can be generated by mutating any one of the 14 CawGG (SEQ ID NO: 54) sites to CCwGG (SEQ ID NO: 3), thereby allowing a total of 2... 14 A number of possible combinations.

[0073] Figure 17 An exemplary generic barcode sequence is shown, which allows for the generation of subsequent barcode sequences by changing one or more of the 14 CawGG (SEQ ID NO: 54) positions to CCwGG. Figure 18 An exemplary barcode sequence and a fragment thereof are shown, wherein only four reference sites are methylated. Exemplary barcode sequences are provided in SEQ ID NO: 39-44. An exemplary barcode sequence is provided in SEQ ID NO: 53.

[0074] In some embodiments, each of the multiple hairpin nucleic acids contains a barcode, wherein the barcode is uniquely associated with the nucleotide sequence of one of the multiple hairpin nucleic acids. In some embodiments, the barcode contains one or more constant sites, wherein each of the multiple constant sites contains the nucleotide sequence CCwGG (SEQ ID NO:3). In some embodiments, the multiple hairpin nucleic acids contain at least two constant sites spaced differently from each other. In some embodiments, the multiple hairpin nucleic acids contain at least two constant sites spaced differently from each other at either end of the fragment. In some embodiments, at least two of the multiple hairpin nucleic acids contain a different number of constant sites from each other. Thus, in some embodiments, at least two of the multiple hairpin nucleic acids, after methylation by DCM methyltransferase in *E. coli*, contain 5-methylcytosine modification patterns that are different from each other.

[0075] ring

[0076] In some embodiments, the first lead-forming sequence 108 and the second lead-forming sequence 109 of the hairpin nucleic acid 100 are connected to each other by a loop 110 to form a continuous polynucleotide sequence. In some embodiments, the hairpin nucleic acid 100 includes a loop 110 located between the two lead-forming sequences. In some embodiments, the size of the loop 110 is in the range of 5 to 100 bases, 5 to 80 bases, 5 to 60 bases, 5 to 40 bases, 5 to 20 bases, 10 to 100 bases, 10 to 80 bases, 10 to 60 bases, 10 to 40 bases, 10 to 20 bases. The range of bases, 20 to 100 bases, 20 to 80 bases, 20 to 60 bases, 20 to 40 bases, 40 to 100 bases, 40 to 80 bases, 40 to 60 bases, 60 to 100 bases, 60 to 80 bases, or 80 to 100 bases.

[0077] In some embodiments, the loop described herein comprises one or more consecutive nucleotides at the 3' end and one or more consecutive nucleotides at the 5' end, wherein the one or more consecutive nucleotides at the 3' end and the one or more consecutive nucleotides at the 5' end are complementary to each other. In some embodiments, the one or more consecutive nucleotides at the 5' end comprise at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 nucleotides. In some embodiments, the one or more consecutive nucleotides at the 3' end comprise at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 nucleotides. In some embodiments, the one or more consecutive nucleotides at the 3' end and the one or more consecutive nucleotides at the 5' end hybridize with each other to produce a blunt end. In some embodiments, the one or more consecutive nucleotides at the 3' end and the one or more consecutive nucleotides at the 5' end hybridize with each other to produce a sticky end. In some embodiments, the sticky end is located at the 3' end or the 5' end. In some embodiments, the sticky end comprises at least 1, at least 2, at least 3, at least 4, or at least 5 single-stranded bases. In some embodiments, the loop further includes a stretching segment of a polynucleotide located between two complementary sequences (e.g., lead-forming sequences) at both ends of a non-complementary contiguous sequence, thus forming a single-stranded hinge. The hinge comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 nucleotides. More preferably, the hinge comprises at least 2, at least 3, at least 4, or at least 5 nucleotides. Even more preferably, the hinge comprises at least 4 nucleotides.

[0078] Connection sequence

[0079] In some embodiments, the intermediate portion of the hairpin nucleic acid described herein includes one or more linker sequences. In some embodiments, the intermediate portion 103 of the hairpin nucleic acid 100 includes a first linker sequence 112, wherein the first linker sequence 112 is located between a first molecule-binding sequence 104 and a first lead-forming sequence 108. In some embodiments, the intermediate portion 103 of the hairpin nucleic acid 100 includes a second linker sequence 113, wherein the second linker sequence 113 is located between a second lead-forming sequence 109 and a second molecule-binding sequence 105. In some embodiments, the intermediate portion 103 of the hairpin nucleic acid 100 includes a first linker sequence 112 and a second linker sequence 113, wherein the first linker sequence 112 is located between the first molecule-binding sequence 104 and the first lead-forming sequence 108, and wherein the second linker sequence 113 is located between the second lead-forming sequence 109 and the second molecule-binding sequence 105. In some embodiments, the first linker sequence 112 and the second linker sequence 113 are not complementary. Therefore, in some embodiments, a first spacer region binding sequence comprising a first linker sequence 112 and a second spacer region binding sequence comprising a second linker sequence 113 form a 4-way link (or holiday link) with the first spacer region polynucleotide, wherein the first spacer region polynucleotide is complementary to the first spacer region binding sequence or a consecutive portion thereof, and wherein the second spacer region polynucleotide is complementary to the second spacer region binding sequence or a consecutive portion thereof. In some embodiments, a first lead-forming sequence 108 and a second lead-forming sequence 109 are located between the first linker sequence 104 and the second linker sequence 105.

[0080] In some embodiments, the linker sequence described herein comprises one or more consecutive nucleotides, comprising at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 nucleotides.

[0081] In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 are complementary to each other. In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 hybridize to form a blunt end. In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 hybridize to form a sticky end. In some embodiments, the sticky end is located at the 3' end or the 5' end. In some embodiments, the sticky end comprises at least one, at least two, at least three, at least four, or at least five single-stranded bases.

[0082] In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 are complementary to each other. In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 hybridize to form blunt ends. In some embodiments, one or more consecutive nucleotides at the 3' end of the first linker sequence 112 and one or more consecutive nucleotides at the 5' end of the second linker sequence 113 hybridize to form sticky ends.

[0083] connector

[0084] In some embodiments, the first end of the hairpin nucleic acid described herein includes a first adaptor. In some embodiments, the first adaptor 106 is configured to anchor the hairpin nucleic acid 100 to a bead. In some embodiments, the first adaptor 106 covalently anchors the hairpin nucleic acid 100 to the bead. In some embodiments, the first adaptor 106 non-covalently anchors the hairpin nucleic acid to the bead. In some embodiments, the first adaptor 106 includes a first nucleotide conjugation group configured to interact with the anchoring molecule of the bead (e.g., covalent interaction, non-covalent interaction, or a combination thereof). In some embodiments, the first nucleotide conjugation group is selected from any of the following: a DBCO group, an azide group, a toluenesulfonyl group, an amino group, a mercapto group, an epoxy group, a thiol group, a hydroxyl group, a chloromethyl group, a streptavidin moiety, or a biotin moiety.

[0085] In some embodiments, the first adaptor 106 is configured to anchor the hairpin nucleic acid 100 to the bead via hybridization. In some embodiments, the first adaptor 106 comprises a polynucleotide sequence engineered to hybridize with the anchoring molecule, wherein the anchoring molecule comprises a polynucleotide sequence complementary to the first adaptor 106. In some embodiments, the polynucleotide sequence of the anchoring molecule comprises at least a portion that is a single-stranded polynucleotide and is configured to hybridize with the first adaptor 106.

[0086] In some embodiments, the second end of the hairpin nucleic acid described herein includes a second adaptor. In some embodiments, the second adaptor 107 is configured to attach the hairpin nucleic acid 100 to a feature portion of the device. In some embodiments, the second adaptor 107 covalently attaches the hairpin nucleic acid to the feature portion. In some embodiments, the second adaptor 107 non-covalently attaches the hairpin nucleic acid to the feature portion. In some embodiments, the second adaptor 107 includes a second nucleotide conjugate molecule configured to interact with the feature portion (e.g., covalent interaction, non-covalent interaction, or a combination thereof). In some embodiments, the second nucleotide conjugate molecule includes a group selected from any one of the following: a DBCO group, an azide group, a toluenesulfonyl group, an amino group, a mercapto group, an epoxy group, a thiol group, a hydroxyl group, a chloromethyl group, a streptavidin moiety, or a biotin moiety.

[0087] Alternatively, in some embodiments, the second adaptor 107 is configured to immobilize the hairpin nucleic acid 100 to a feature portion of the device via hybridization. In some embodiments, the second adaptor 107 comprises a polynucleotide sequence engineered to hybridize with the feature portion, wherein the feature portion comprises a polynucleotide sequence complementary to the second adaptor 107. In some embodiments, the polynucleotide sequence of the feature portion comprises at least a portion that is single-stranded and configured to hybridize with the second adaptor 107.

[0088] Exemplary sequences of the adaptor are provided in SEQ ID NO: 11 and 36 for bead-anchored oligonucleotides and SEQ ID NO: 14 and 37 for surface-anchored oligonucleotides.

[0089] Spacer polynucleotides

[0090] In some embodiments, the system described herein comprises one or more spacer polynucleotides. In some embodiments, the spacer polynucleotides described herein comprise a polynucleotide chain complementary to a first spacer binding sequence or a second spacer binding sequence. In some embodiments, the spacer polynucleotides described herein comprise a polynucleotide chain complementary to both a first spacer binding sequence and a loop. In some embodiments, the spacer polynucleotides described herein comprise a polynucleotide chain complementary to both a loop and a second spacer binding sequence. In some embodiments, the spacer polynucleotides described herein comprise a polynucleotide chain complementary to both a first spacer binding sequence, a loop, and a second spacer binding sequence. In some embodiments, the spacer polynucleotide prevents hairpin formation after hybridization with the complementary spacer binding sequence. Therefore, in some embodiments, the spacer polynucleotide prevents hybridization between the first lead-forming sequence and the second lead-forming sequence after hybridization with the complementary spacer binding sequence. In some embodiments, the first spacer polynucleotide and the second spacer polynucleotide hybridize together to form a Y-shape through the complementary sequence on each polynucleotide.

[0091] In some embodiments, the spacer polynucleotides described herein comprise Y-shaped (or substantially Y-shaped) spacer polynucleotides, wherein the Y-shaped spacer polynucleotide comprises two spacer polynucleotide chains (e.g., a first spacer polynucleotide and a second spacer polynucleotide), each spacer polynucleotide chain comprising a lead-forming sequence binding region and a linker sequence binding region. In some embodiments, the lead-forming sequence binding regions of the two spacer polynucleotides hybridize with each other to form a Y-shaped spacer polynucleotide with blunt ends. In some embodiments, the lead-forming sequence binding regions of the two spacer polynucleotides hybridize with each other to form a Y-shaped spacer polynucleotide with sticky ends. In some embodiments, the sticky ends are located at the 3' end or the 5' end. In some embodiments, the sticky ends comprise at least one, at least two, at least three, at least four, or at least five single-stranded bases. In some embodiments, the lead-forming sequence binding regions of the two spacer polynucleotides are complementary to the two lead-forming sequences of the hairpin nucleic acid. In some embodiments, the linker sequence binding regions of the two spacer polynucleotides are complementary to the two linker sequences of the hairpin nucleic acid. In some embodiments, a portion of the linker sequence binding region of the two spacer polynucleotide chains is complementary to portions of the two linker sequences of the hairpin nucleic acid. Therefore, in some embodiments, the two spacer polynucleotides and the two linker sequences form a 4-way link (or holiday link). Alternatively, in some embodiments, the first and second spacer polynucleotides form a Y-shaped spacer polynucleotide that forms a 4-way link (or holiday link) after hybridization with the first and second linker sequences of the hairpin nucleic acid 100, respectively. In some embodiments, the lead-forming sequence binding regions of the two spacer polynucleotides are connected to each other via spacer loops complementary to the loops of the hairpin nucleic acid 100. Alternatively, in some embodiments, the spacer polynucleotides comprise a polynucleotide sequence complementary to the middle portion 103 of the hairpin nucleic acid 100. In some embodiments, the spacer polynucleotides described herein comprise the nucleotide sequence of SEQ ID NO: 12 or 13.

[0092] Figure 5 An exemplary structure of spacer polynucleotide 500 is shown. As shown, spacer polynucleotide 500 includes a first spacer polynucleotide chain 501 and a second spacer polynucleotide chain 502. The first spacer polynucleotide chain 501 includes a first linker sequence binding region 505 and a first lead-forming sequence binding region 503. The second spacer polynucleotide chain 502 includes a second linker sequence binding region 506 and a second lead-forming sequence binding region 504. Furthermore, as shown, the 5' end of the first spacer polynucleotide chain 501 and the 3' end of the second spacer polynucleotide chain 502 are connected to each other via a spacer loop 507.

[0093] Figure 6Two non-limiting exemplary spacer polynucleotides are shown that can be used to prepare Y-shaped spacer polynucleotides: (a) a first spacer polynucleotide chain 501 (5' to 3') comprising a first linker sequence binding region 505 and a first lead-forming sequence binding region 503, and (b) a second spacer polynucleotide chain 502 (5' to 3') comprising a second linker sequence binding region 504 and a second lead-forming sequence binding region 506.

[0094] Figure 7 A non-limiting exemplary system comprising a holiday connection formed between a nucleic acid scaffold and a spacer polynucleotide is shown. The holiday connection can be eliminated by applying sufficient force because the two lead sequences of molecule 100 and spacer molecule 500 are complementary. In some embodiments, sufficient force refers to a force in the range of 5 pN to 10 pN.

[0095] Candidate molecules

[0096] In some embodiments, the candidate molecules described herein relate to or cause the etiology of a disease. In some embodiments, the candidate molecules relate to the prevention or treatment of a disease. In some embodiments, the candidate molecules are derived from viruses, bacteria, fungi, or mammalian cells. In some embodiments, the candidate molecules are isolated from cells separated from cancerous or pathological tissues, such as tumors or amyloid plaques.

[0097] In some embodiments, the candidate molecule includes small molecules. In some embodiments, the small molecule includes molecules with a molecular weight ranging from 50 Daltons to 20,000 Daltons. In some embodiments, the candidate molecule includes a protein of interest. In some embodiments, the protein of interest includes a polypeptide having at least two amino acids. In some embodiments, the candidate molecule includes a nucleic acid of interest. In some embodiments, the nucleic acid of interest includes DNA, RNA, or combinations thereof. In some embodiments, the nucleic acid of interest includes at least one conformational structure. The conformational structure of the nucleic acid (or target nucleic acid) refers to a secondary or tertiary conformation, such as at least one selected from RNA hairpins, P-shaped RNA, Y-shaped RNA, candy-shaped RNA, and combinations thereof. In some embodiments, the nucleic acid of interest includes an aptamer. In some embodiments, the aptamer includes a DNA aptamer, an RNA aptamer, an XNA aptamer, or a combination thereof. In some embodiments, the nucleic acid of interest includes a non-coding region of RNA. In some embodiments, the nucleic acid of interest includes a coding region of RNA.

[0098] In some embodiments, the system described herein includes candidate molecules or a library of candidate molecules. In some embodiments, the library of candidate molecules includes analogs and / or chemically modified analogs of selected members of the library.

[0099] In some embodiments, the candidate molecule disclosed herein is anchored to a hairpin nucleic acid 100. In some embodiments, the candidate molecule is covalently anchored to the hairpin nucleic acid 100. In some embodiments, the candidate molecule is non-covalently anchored to the hairpin nucleic acid 100. In some embodiments, the candidate molecule is anchored to the hairpin nucleic acid 100 via hybridization, wherein the candidate molecule is linked to a gel sequence complementary to the molecular binding sequence of the hairpin nucleic acid 100. In some embodiments, the first gel sequence and the second gel sequence are different from each other. In some embodiments, the gel sequence described herein contains an active group configured to link with the candidate molecule. Alternatively, in some embodiments, the gel sequence described herein contains a first active group, and the candidate molecule contains a second active group, wherein the first and second active groups interact with each other to link the gel sequence and the candidate molecule.

[0100] In some implementations, the gel sequence described herein comprises a polynucleotide complementary to a small hairpin nucleic acid as described herein. Figure 19C A graphical representation of a gel sequence containing a small hairpin nucleic acid complementary to a molecularly binding sequence is shown.

[0101] In some embodiments, the gel sequence comprises a single-stranded polynucleotide sequence 708. In some embodiments, the size of the gel sequence is in the range of 5 to 100 bases, 5 to 80 bases, 5 to 60 bases, 5 to 40 bases, 5 to 20 bases, 10 to 100 bases, 10 to 80 bases, 10 to 60 bases, 10 to 40 bases, 10 to 20 bases. The range of bases, 20 to 100 bases, 20 to 80 bases, 20 to 60 bases, 20 to 40 bases, 40 to 100 bases, 40 to 80 bases, 40 to 60 bases, 60 to 100 bases, 60 to 80 bases, or 80 to 100 bases.

[0102] The system described herein includes a first candidate molecule and a second candidate molecule. In some embodiments, the first candidate molecule is anchored to a first molecule-binding sequence 104 of the hairpin nucleic acid 100 via a first gel sequence. In some embodiments, the second candidate molecule is anchored to a second molecule-binding sequence 105 of the hairpin nucleic acid 100 via a second gel sequence. Furthermore, in some embodiments, the system described herein also includes a third candidate molecule, wherein the third candidate molecule promotes the interaction between the first candidate molecule and the second candidate molecule. Alternatively, in some embodiments, the system described herein also includes a third candidate molecule, wherein the third candidate molecule prevents the interaction between the first candidate molecule and the second candidate molecule. In some embodiments, the third candidate molecule interacts with the first candidate molecule, the second candidate molecule, or both. Therefore, in some embodiments, the interaction between the third molecule and the first molecule promotes / prevents the interaction between the first molecule and the second molecule. In some embodiments, the interaction between the first molecule, the second molecule, and the third molecule promotes / prevents the interaction between the first molecule and the second molecule.

[0103] beads

[0104] In some embodiments, the beads disclosed herein are configured to anchor the hairpin nucleic acid 100 described herein via anchoring molecules. In some embodiments, the anchoring molecules are covalently attached to the beads. In some embodiments, the beads are made of a non-conductive material (e.g., polymer, silicon, glass, resin, or combinations thereof).

[0105] In some embodiments, the beads include diameters ranging from 0.1 μm to 10 μm, 0.3 μm to 10 μm, 0.5 μm to 10 μm, 1 μm to 10 μm, 2 μm to 10 μm, 5 μm to 10 μm, 0.1 μm to 5 μm, 0.3 μm to 5 μm, 0.5 μm to 5 μm, 1 μm to 5 μm, 2 μm to 5 μm, 0.1 μm to 2 μm, 0.3 μm to 2 μm, 0.5 μm to 2 μm, 1 μm to 2 μm, 0.1 μm to 1 μm, 0.3 μm to 1 μm, 0.5 μm to 1 μm, 0.1 μm to 0.5 μm, 0.3 μm to 0.5 μm, or 0.1 μm to 0.3 μm. In some implementations, the diameter of the magnetic beads is 0.3µm, 0.5µm, 1.04µm, 2.8µm, or 5.5µm.

[0106] In some embodiments, the beads described herein include magnetic beads. In some embodiments, the magnetic beads (e.g., magnetic beads "MyOne", manufactured by Invitrogen, having a diameter of 1.04 μm; M270 manufactured by Invitrogen, having a diameter of 2.8 μm; M450 manufactured by Invitrogen, having a diameter of 5.5 μm; Ademtech 500 manufactured by Ademtech, having a diameter of 0.5 μm; Ademtech 300 manufactured by Ademtech, having a diameter of 0.3 μm) are configured to anchor the first end of the hairpin nucleic acid described herein.

[0107] The anchoring molecule described herein is configured to anchor the hairpin nucleic acid (e.g., DNA, RNA, or a combination thereof) described herein to a bead. In some embodiments, the hairpin nucleic acid 100 is anchored to the bead by the anchoring molecule. Alternatively, in some embodiments, the hairpin nucleic acid 100 is anchored to the anchoring molecule through an interaction (e.g., covalent or non-covalent interaction). In some embodiments, the anchoring molecule is configured to form a covalent bond with a first adaptor 106 at the first end of the hairpin nucleic acid. In some embodiments, the anchoring molecule comprises a group selected from the group DBCO, azide, toluenesulfonyl, amino, mercapto, epoxy, thiol, hydroxy, chloromethyl, streptavidin moiety, or biotin moiety. Therefore, in some embodiments, the anchoring molecule and the first linker 106 comprise a combination of groups that form covalent bonds with each other, wherein the combination of groups comprises: (a) a DBCO group and an azide group; (b) a toluenesulfonyl group and an amino group; (c) a toluenesulfonyl group and a mercapto 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. Therefore, in some embodiments, the first linker 106 comprises a DBCO group, and the bead comprises an azide group. Conversely, in some embodiments, the first linker 106 comprises an azide group, and the bead comprises a DBCO group. In some embodiments, the two groups having a non-covalent interaction therebetween comprise a biotin moiety and a streptavidin moiety. Therefore, in some embodiments, the first linker 106 comprises a biotin moiety, and the surface of the device comprises a streptavidin moiety. Conversely, in some embodiments, the first linker 106 comprises a streptavidin moiety, and the bead comprises a biotin moiety.

[0108] Alternatively, the anchoring molecule described herein is configured to anchor the hairpin nucleic acid described herein to a bead via hybridization. In some embodiments, the hairpin nucleic acid is anchored to the bead via hybridization between the polynucleotide sequence of the anchoring molecule and the polynucleotide sequence of the first adaptor 106, wherein the polynucleotide sequence of the anchoring molecule and the polynucleotide sequence of the first adaptor 106 are complementary to each other, and wherein at least a portion of the polynucleotide sequence of the anchoring molecule is single-stranded. In some embodiments, the single-stranded portion of the polynucleotide sequence of the anchoring molecule is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the polynucleotide sequence of the first adaptor 106. In some embodiments, the single-stranded portion of the polynucleotide sequence of the anchoring molecule comprises 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.

[0109] Device

[0110] The apparatus disclosed herein includes an actuator, a sensor, and a chamber. In some embodiments, the actuator is adapted to move a bead relative to a surface of the apparatus in a predetermined direction. In some embodiments, the sensor is adapted to measure the distance between the paramagnetic bead and the surface. In some embodiments, the chamber includes an axis extending along the predetermined direction, a bead, and a bottom surface. In some embodiments, the bottom surface includes one or more feature portions. In some embodiments, one or more activated molecules are covalently attached to the feature portions.

[0111] In some embodiments, the apparatus described herein includes a force application mechanism (e.g., an actuator). In some embodiments, the actuator described herein is configured to generate a force. Thus, in some embodiments, the actuator is configured to use a force to move a bead within a chamber. In some embodiments, the force applied to the bead is constant. In some embodiments, the force applied to the bead increases (ramp). The change in the position of the bead within the chamber is measured using sensors of the apparatus. In some embodiments, the force is magnetic. In some embodiments, the force is the force of laser radiation pressure.

[0112] In some embodiments, the actuator described herein is configured to generate a magnetic force. Therefore, in some embodiments, the actuator is configured to use the magnetic force to move a bead within a chamber. In some embodiments, the magnetic force applied to the bead is constant. In some embodiments, the magnetic force applied to the bead increases (ramp). The change in the position of the bead within the chamber is measured using the device's sensors.

[0113] In some embodiments, the actuator described herein is an optical tweezers. Therefore, in some embodiments, the optical tweezers are configured to capture a bead in a chamber using laser radiation pressure. In some embodiments, the laser radiation pressure applied to the bead is constant. In some embodiments, the laser radiation pressure applied to the bead is increased (ramp). Changes in the position of the bead in the chamber are measured using sensors of the device.

[0114] In some embodiments, the sensor includes a camera (e.g., a CMOS camera) capable of collecting photons reflected from the paramagnetic bead. In some embodiments, the sensor includes a CMOS sensor capable of measuring the impedance from the feature area caused by the up-and-down movement of the bead.

[0115] In some embodiments, the device described herein includes: (a) a surface (e.g., silicon, glass, a non-conductive polymer, or resin) configured to immobilize the second end of the hairpin nucleic acid described herein; (c) an actuator adapted to move the bead described herein relative to the surface of the device in one direction of motion; (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 conductive solution contains 10 -7 S / cm to 10 1 Between S / cm or 10 -3 S / cm to 10 -2 Conductivity between S / cm, wherein the sensor is adapted to measure the impedance of the chamber, which is a function of the distance between the bead and the surface.

[0116] The apparatus described herein may include: a) an objective lens for collecting light 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 such that a first portion of the radiation diffused by the object and a second portion of the radiation diffused by the object pass through the first aperture and the second aperture, respectively, while blocking a portion of radiation emitted by a light source that is not diffused by the object; and c) a detector adapted to generate an image including a first spot and a second spot, the first spot and the second spot representing a first portion and a second portion of the radiation impacting 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 spot and the second spot relative to each other along the second axis.

[0117] In some embodiments, the sensor described herein may include: a main electrode located at the top of the chamber and in contact with a conductive solution, the electrode being applied a known potential; a second electrode located at the bottom of the chamber and carrying a surface capable of attaching molecules; and electronic circuitry adapted to measure the current flowing between the electrodes, the electronic circuitry including: a current-voltage amplifier connected to the second electrode; a voltmeter adapted to measure the output voltage of the current-voltage amplifier; and computational circuitry adapted to calculate the impedance of the well from the measured voltage. In some embodiments, the sensor includes a camera CMOS configured to collect photons reflected from the beads. In some embodiments, the sensor includes a CMOS configured to measure the impedance from a feature region caused by the up-and-down movement of the beads.

[0118] In some embodiments, the actuators described herein include at least one pair of actuators configured to control the movement of a bead along an X-axis, Y-axis, Z-axis, or a combination thereof. In some embodiments, the actuators apply a constant force to the bead, allowing control of the bead's movement. In some embodiments, the actuators apply a variable force (e.g., an increasing or decreasing force) to the bead, allowing control of the bead's movement. In some embodiments, the force applied to the bead by the actuator is at least 0.01 pN, at least 0.1 pN, at least 1 pN, at least 2 pN, at least 3 pN, at least 5 pN, at least 10 pN, at least 20 pN, at least 40 pN, at least 60 pN, or at least 95 pN. In some embodiments, the force applied to the bead by the actuator does not exceed 100 pN. In some embodiments, the force required to control the movement of the bead in the presence of a candidate molecule is compared with the force required to control the movement of the bead in the absence of a candidate molecule. Examples of the force applied to the beads by the actuator can be 0.1 pN to 35 pN or greater and up to 100 pN. In some embodiments, the force applied to the beads by the actuator can be 0.01 pN to 35 pN or greater and up to 100 pN.

[0119] In some embodiments, the actuator described herein includes at least a pair of magnets (e.g., permanent magnets, soft magnets, or combinations thereof) configured to control translational movement along the XX-axis. In some embodiments, the actuator includes two permanent magnets located equidistant from the XX-axis and with their magnetic poles aligned perpendicular to the XX-axis, the north pole of one magnet facing the south pole of the other. In some embodiments, the force of the magnets relative to the beads is constant. In some cases, the force of the magnets relative to the beads is increased or decreased by moving the permanent magnets relative to the beads. In some embodiments, the force of the magnets generated on the beads is at least 0.01 pN, at least 0.1 pN, at least 1 pN, at least 2 pN, at least 3 pN, at least 5 pN, at least 10 pN, at least 20 pN, at least 40 pN, at least 60 pN, or at least 95 pN. In some embodiments, the force of the magnets generated on the beads does not exceed 100 pN. The bead positions in the presence of candidate molecules are compared with the bead positions without candidate molecules under the same force. Alternatively, in some embodiments, the force of the magnet required to hold the bead in a specific position in the presence of the candidate molecule is compared with the force required to hold the bead in the same position in the absence of the candidate molecule. An example of the force of the magnet relative to the bead may be 0.1 pN to 35 pN or greater and up to 100 pN. In some embodiments, the force applied to the bead by the actuator may be 0.01 pN to 35 pN or greater and up to 100 pN. In some embodiments, at least one pair of magnets includes more than one pair of magnets, more than five pairs of magnets, more than ten pairs of magnets, more than twenty pairs of magnets, more than fifty pairs of magnets, or more than one hundred pairs of magnets. In some embodiments, at least one pair of magnets includes 1 to 500 pairs of magnets, 1 to 400 pairs of magnets, 1 to 300 pairs of magnets, 1 to 200 pairs of magnets, 1 to 100 pairs of magnets, 1 to 50 pairs of magnets, 1 to 10 pairs of magnets, 1 to 5 pairs of magnets, 50 to 500 pairs of magnets, 50 to 400 pairs of magnets, 50 to 300 pairs of magnets, and 50 to 200 pairs of magnets. Magnets, 50 to 100 pairs of magnets, 100 to 500 pairs of magnets, 100 to 400 pairs of magnets, 100 to 300 pairs of magnets, 100 to 200 pairs of magnets, 200 to 500 pairs of magnets, 200 to 400 pairs of magnets, 200 to 300 pairs of magnets, 300 to 500 pairs of magnets, 300 to 400 pairs of magnets or 400 to 500 pairs of magnets.

[0120] As disclosed herein, a feature refers to a binding portion on the surface of the device described herein. Therefore, in some embodiments, the feature is configured to anchor a hairpin nucleic acid (e.g., DNA, RNA, or a combination thereof) described herein to the surface of the device. The anchoring device is fixed to the surface of the device by one side and does not prevent movement. Alternatively, in some embodiments, the hairpin nucleic acid is anchored to the surface of the device through interaction with the feature (e.g., covalent or non-covalent interaction). In some embodiments, the feature is configured to form a covalent bond with a second adaptor 107 at the second end of the hairpin nucleic acid. In some embodiments, the feature comprises a group selected from any of the following: a DBCO group, an azide group, a toluenesulfonyl group, an amino group, a mercapto group, an epoxy group, a thiol group, a hydroxyl group, a chloromethyl group, a streptavidin portion, or a biotin portion. Therefore, in some embodiments, the feature portion and the second adapter 107 comprise a combination of groups that form covalent bonds with each other, wherein the combination of groups comprises: (a) a DBCO group and an azide group; (b) a toluenesulfonyl group and an amino group; (c) a toluenesulfonyl group and a mercapto 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. Therefore, in some embodiments, the second adapter 107 comprises a DBCO group, and the feature portion comprises an azide group. Conversely, in some embodiments, the second adapter 107 comprises an azide group, and the feature portion comprises a DBCO group. In some embodiments, the two groups having a non-covalent interaction therebetween comprise a biotin moiety and a streptavidin moiety. Therefore, in some embodiments, the second adapter 107 comprises a biotin moiety, and the feature portion comprises a streptavidin moiety. Conversely, in some embodiments, the second adapter 107 comprises a streptavidin moiety, and the feature portion comprises a biotin moiety.

[0121] In some embodiments, the feature portion of the device surface is configured to immobilize the hairpin nucleic acid described herein via hybridization. In some embodiments, the hairpin nucleic acid is immobilized to the surface of the device by hybridization between the polynucleotide sequence of the feature portion and the polynucleotide sequence of the second adaptor 107, wherein the polynucleotide sequence of the feature portion and the polynucleotide sequence of the second adaptor 107 are complementary to each other, and wherein at least a portion of the polynucleotide sequence of the feature portion is single-stranded. In some embodiments, the single-stranded portion of the polynucleotide sequence of the feature portion is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the polynucleotide sequence of the second adaptor 107. In some embodiments, the single-stranded portion of the polynucleotide sequence of the feature portion comprises 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.

[0122] As disclosed herein, the device may include a plurality of features. For example, the device may include at least 1, at least 10, at least 100, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10000, at least 20000, at least 30000, at least 40000, at least 50000, at least 60000, at least 70000, at least 80000, at least 90000, at least 100000, at least 500000, at least 100000, at least 2000000, at least 500000, or more than 10,000000 features. Therefore, in some embodiments, the feature portions as described herein represent at least 1,000 feature portions. Therefore, in some embodiments, the feature portions as described herein represent at least 2,000,000 feature portions. Therefore, single binding experiments can be performed simultaneously on a large number of individual molecules.

[0123] In some embodiments, the feature portions are covalently and / or non-covalently attached to the surface of the device. Covalent attachment of the feature portions to the device surface can be performed by a technician (e.g., esterification, click chemistry). In some embodiments, the surface of the device includes at least two feature portions. In some embodiments, at least two feature portions are identical relative to each other. In some embodiments, at least two feature portions are different relative to each other. In some embodiments, having different feature portions facilitates anchoring different hairpin nucleic acids at specific locations within the device. This allows for rapid mapping of different hairpin nucleic acids within the device.

[0124] Nucleic acid scaffold

[0125] This document discloses a nucleic acid scaffold. In some embodiments, the nucleic acid scaffold comprises a hairpin nucleic acid 100, a first spacer region polynucleotide hybridized to a first spacer region binding sequence of the hairpin nucleic acid 100, and a second spacer region polynucleotide hybridized to a second spacer region binding sequence of the hairpin nucleic acid 100. In some embodiments, the nucleic acid scaffold is anchored to a bead by attaching a first terminal 101 of the hairpin nucleic acid 100 to the bead. In some embodiments, the nucleic acid scaffold is fixed to a feature portion on the surface of a device by attaching a second terminal 102 of the hairpin nucleic acid 100 to a feature portion. In some embodiments, the spacer region polynucleotide hybridizes to a first linker sequence 112 and a second linker sequence 113 of the nucleic acid scaffold. In some embodiments, the spacer region polynucleotide prevents the formation of a hairpin structure and forms the nucleic acid scaffold when it binds to the first linker-forming sequence 112, the second linker-forming sequence 113, the first lead-forming sequence 108, the second lead-forming sequence 109, and the loop 110. In some embodiments, the length of the nucleic acid scaffold is a reference elongation length. In some embodiments, a reference elongation length is determined when a magnetic force is applied to the magnetic bead, wherein the nucleic acid scaffold is fixed between the magnetic bead and the surface of the device. In some embodiments, the reference elongation length is determined under a magnetic force of 0.2 pN applied to the magnetic bead, wherein the nucleic acid scaffold is fixed between the magnetic bead and the surface of the device. In some embodiments, the reference elongation length is adjusted by changing the size of the hairpin nucleic acid.

[0126] In some embodiments, when the first candidate molecule and the second candidate molecule are respectively located, the first molecule binding sequence 104 and the second molecule binding sequence 105 of the nucleic acid scaffold form a screening nucleic acid scaffold. In some embodiments, when 0.1pN to 50pN, 0.1pN to 40pN, 0.1pN to 30pN, 0.1pN to 20pN, 0.1pN to 10pN, 0.2pN to 50pN, 0.2pN to 40pN, 0.2pN to 30pN, 0.2pN to 20pN, 0.2pN to 10pN, 0.5pN to 50pN, 0.5pN to 40pN are applied to the magnetic beads... When a magnetic force of N, 0.5pN to 30pN, 0.5pN to 20pN, 0.5pN to 10pN, 1pN to 50pN, 1pN to 40pN, 1pN to 30pN, 1pN to 20pN, or 1pN to 10pN, 10pN to 50pN, 10pN to 40pN, 10pN to 30pN, or 10pN to 20pN is applied, the elongation length of the screening nucleic acid scaffold is less than the reference elongation length. In some embodiments, when a magnetic force is applied to the magnetic beads, the elongation length of the screening nucleic acid scaffold is less than the reference elongation length. In some embodiments, when a magnetic force of 0.2pN is applied to the magnetic beads, the elongation length of the screening nucleic acid scaffold is less than the reference elongation length.

[0127] In some embodiments, the screening nucleic acid scaffold as described herein is incubated together with a third candidate molecule, resulting in a change in the elongation length of the screening nucleic acid scaffold or a change in the force required to achieve the same elongation length as a reference elongation length. In some embodiments, the third candidate molecule binds to the first candidate molecule, the second candidate molecule, or both. In some embodiments, the third candidate molecule promotes the binding of the first and second candidate molecules, resulting in a reduced elongation length of the screening nucleic acid scaffold or requiring greater force to achieve the same elongation length as the reference elongation length. Conversely, in some embodiments, the third candidate molecule inhibits the binding of the first and second candidate molecules, resulting in an elongation length of the screening nucleic acid scaffold similar to the reference elongation length without molecules, or requiring less force to achieve the same elongation length as the reference elongation length.

[0128] Conductive solution

[0129] In some embodiments, the system described herein includes a conductive solution disposed within 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 bead and a feature (e.g., a surface). In some embodiments, the conductive solution may have a conductivity of 10. -7 S / cm to 10 1 Between S / cm or 10 -3 S / cm to 10 -2 Conductivity between S / cm.

[0130] Reagent test kit

[0131] This document also discloses a kit comprising one or more components of the system disclosed herein. In some embodiments, one or more components comprise the hairpin nucleic acid described herein, the spacer polynucleotide described herein, the candidate molecule described herein, the beads described herein, and the gel sequence described herein. In some embodiments, one or more components comprise a hairpin nucleic acid having a barcode containing at least one modified base described herein, an antibody specific to the modified base of the barcode, the spacer polynucleotide described herein, the candidate molecule described herein, the beads described herein, and the gel sequence described herein. In some embodiments, the kit is configured to form a nucleic acid scaffold, wherein the nucleic acid scaffold comprises a hairpin nucleic acid 100 and a spacer polynucleotide. In some embodiments, the kit is configured to form a nucleic acid scaffold, wherein the nucleic acid scaffold does not contain a double-stranded DNA molecule comprising a first double-stranded DNA molecule (1) connected to a second double-stranded DNA molecule (2) by a tie containing double-stranded DNA, wherein the tie: (i) is a nucleotide attached to the first double-stranded DNA molecule (1) by at least one covalent bond, and (ii) is a nucleotide attached to the second double-stranded DNA molecule (2) by at least one covalent bond. In some embodiments, the nucleic acid scaffold further comprises one or more candidate molecules described herein. In some embodiments, the kit is configured to determine binding interactions between two or more candidate molecules. In some embodiments, the kit is configured to screen for candidate molecules that have binding interactions with one or more candidate molecules from a candidate molecule library, wherein the binding interaction force is in the range of 0.01 pN to 100 pN or 0.1 pN to 100 pN. In some embodiments, the kit is configured to screen for candidate molecules having binding interaction forces in the range of 0.01 pN to 5 pN or 0.1 pN to 5 pN.

[0132] In some embodiments, the kit may include a container and instructions for use. In some embodiments, the kit may also include a candidate molecular library. In some embodiments, the candidate molecular library includes small molecule libraries, protein libraries, antibody libraries, aptamer libraries, and advantageously libraries containing binding molecules. In some embodiments, the kit may also include a buffer containing a monovalent cation in the range of 1 nM to 1 M. In some embodiments, the monovalent cation includes Na+. + K + Li + Or a combination thereof. In some embodiments, the buffer contains approximately 500 mM Na. + Approximately 150 mM Na + Approximately 137 mM Na + Approximately 10mM Na + Approximately 1 mM Na + Approximately 500 μM Na+ Approximately 100 μM Na + Approximately 10 μM Na + Approximately 1 μM Na + Approximately 500 nM Na + Approximately 100 nM Na + Approximately 10 nM Na + Or approximately 1 nM Na + In some implementations, the buffer solution contains approximately 150 mM Na. + In some implementations, the buffer solution contains approximately 137 mM Na. + In some implementations, the buffer solution contains approximately 500 mM K. + Approximately 150mM K + Approximately 137mM K + Approximately 13mM K + Approximately 10 mm K + Approximately 1 mm K + Approximately 500 μM K + Approximately 100 μM K + Approximately 10 μM K + Approximately 1 μM K + Approximately 500 nM K + Approximately 100 nM K + Approximately 10 nM K + or approximately 1 nM K + In some implementations, the buffer solution contains approximately 150 mM K. + In some implementations, the buffer solution contains approximately 13 mM K. + In some implementations, the buffer solution contains approximately 137 mM Na. + and approximately 13mM K + In some embodiments, the kit further includes a buffer containing divalent cations in the range of 1 μM to 1 mM. In some embodiments, the divalent cations include Mg. 2+ Mn 2+ Zn 2+ Fe 2+ Cu 2+ Cd 2 + Or a combination thereof. In some embodiments, the buffer solution contains less than 10 mM Mg. 2+ Less than 5mM Mg 2+ Favorably >0.01mM Mg 2+ And less than 10 mM Mg 2+ More favorable 1mM Mg 2+ .

[0133] Methods for preparing hairpin nucleic acids

[0134] This document discloses a method for preparing hairpin nucleic acid 100, wherein the hairpin nucleic acid 100 comprises a first terminal 101 as described herein, a middle portion 103 as described herein, and a second terminal 102 as described herein. In some embodiments, the method includes: (a) providing a first hairpin nucleic acid precursor comprising a first terminal and a second terminal of the first hairpin nucleic acid precursor, wherein the first hairpin nucleic acid precursor is a double-stranded polynucleotide, wherein each strand of the first hairpin nucleic acid precursor comprises a lead-forming sequence; (b) ligating a loop to the first terminal of the first hairpin nucleic acid precursor to form a second hairpin nucleic acid precursor; and (c) ligating a first Y-shaped forming polynucleotide encoding one or more of the first terminal 101 and a first linker sequence 112 of the hairpin nucleic acid 100, and a second Y-shaped forming polynucleotide encoding one or more of the second terminal 102 and a second linker sequence 113 of the hairpin nucleic acid 100 to the second hairpin nucleic acid precursor to form hairpin nucleic acid 100. In some embodiments, the ligation product is purified / recovered by any suitable method (e.g., gel purification).

[0135] In some embodiments, the first hairpin nucleic acid precursor is chemically synthesized. In some embodiments, the first hairpin nucleic acid precursor is synthesized in vivo (e.g., in *E. coli*). For example, the in vivo synthesis of the first hairpin nucleic acid precursor includes: (a) cloning the first hairpin nucleic acid into a plasmid; (b) propagating the plasmid in a host (e.g., *E. coli*); (c) recovering the plasmid from the host; and (d) digesting the recovered plasmid with a restriction enzyme to form the first hairpin nucleic acid precursor.

[0136] In some embodiments, the first hairpin nucleic acid precursor includes a lead-forming sequence. Therefore, in some embodiments, the first hairpin nucleic acid precursor also includes a barcode. In some embodiments, the barcode includes one or more nucleotides that have undergone epigenetic modification. For example, in some embodiments, the barcode includes one or more cytosines that have undergone epigenetic modification (e.g., methylation). In some embodiments, the barcode includes one or more constant sites spaced apart within the first hairpin nucleic acid precursor (e.g., CCwGG (SEQ ID NO: 3)). Therefore, in some embodiments, the first hairpin nucleic acid precursor includes one or more methylated cytosines when it multiplies in *E. coli*. In some embodiments, the barcode includes two constant sites spaced apart within the first hairpin nucleic acid precursor (e.g., CCwGG (SEQ ID NO: 3)). In some embodiments, the two constant sites present in the first hairpin nucleic acid precursor are spaced 18 and 10 bases apart at either end of the first hairpin nucleic acid precursor. Alternatively, in some implementations, two constant sites present in the first hairpin nucleic acid precursor are spaced 20 and 10 bases apart at either end of the first hairpin nucleic acid precursor. Figure 8 A to Figure 8 Figure D illustrates a graphical representation of a method for identifying hairpin nucleic acids. As shown, this graphical representation provides a method for identifying methylated cytosine (5mC) using an anti-5mC antibody. The number of 5mC sites detected per cycle depends on the concentration of the anti-5mC antibody. In some embodiments, the antibody is injected at concentrations that allow detection of 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, or 5 to 6 methylated cytosine sites per cycle in the universal barcode sequence of the hairpin nucleic acid. Therefore, this cycle can be repeated multiple times until all positions of the universal barcode sequence with 5mC modification have been extracted. In some implementations, the universal barcode sequence of the hairpin nucleic acid described herein has at least 18, at least 16, at least 14, at least 12, at least 10, at least 8 sites, at least 6 sites, at least 4 sites, at least two sites, or at least one methylated cytosine. Figure 9 and Figure 21 Exemplary barcode sequences with constant sites at different locations are shown. In some embodiments, other barcode sequences containing alternative methylated bases can be prepared in various hosts and used with specific antibodies. These are part of the kits provided herein.

[0137] Methods for preparing spacer polynucleotides

[0138] This document discloses a method for preparing spacer polynucleotide hairpins as described herein. In some embodiments, the method includes: (a) providing a first hairpin nucleic acid precursor comprising a first end and a second end, wherein the first hairpin nucleic acid precursor is a double-stranded polynucleotide, wherein each strand of the first hairpin nucleic acid precursor comprises a lead-forming sequence; and (b) linking a first Y-shaped forming polynucleotide encoding a sequence complementary to a first binding sequence 112 and a second Y-shaped forming polynucleotide encoding a sequence complementary to a second binding sequence 113 to the first end of the double-stranded first hairpin nucleic acid precursor to form a spacer polynucleotide. In some embodiments, the spacer polynucleotide comprises blunt ends or sticky ends. In some embodiments, the method further includes linking a spacer loop to the blunt or sticky ends. In some embodiments, the Y-shaped spacer polynucleotide is complementary to the Y-shaped hairpin nucleic acid.

[0139] Methods for preparing nucleic acid scaffolds

[0140] This document discloses methods and systems for preparing nucleic acid scaffolds. In some embodiments, the method includes: (a) providing a hairpin nucleic acid 100 as described herein, wherein the hairpin nucleic acid 100 is a continuous polynucleotide sequence; (b) attaching beads to a first end 101 of the hairpin nucleic acid 100; and (c) attaching a second end 102 of the hairpin nucleic acid 100 to a bottom surface of a device; (d) hybridizing a first spacer polynucleotide with a first spacer binding sequence of the hairpin nucleic acid 100, wherein the first spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of the first spacer binding sequence; and (d) hybridizing a second spacer polynucleotide with a second spacer binding sequence of the hairpin nucleic acid 100, wherein the second spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of the second spacer binding sequence. In some embodiments, a first spacer polynucleotide and a second spacer polynucleotide are provided together, wherein the first spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of the first spacer binding sequence of the hairpin nucleic acid 100, wherein the second spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of the second spacer binding sequence of the hairpin nucleic acid 100, and wherein the first spacer polynucleotide hybridizes with the first spacer binding sequence, and the second spacer polynucleotide hybridizes with the second spacer binding sequence. In some embodiments, the scaffold described herein excludes double-stranded DNA molecules comprising a first double-stranded DNA molecule linked to a second double-stranded DNA molecule (2) by a tie strand comprising double-stranded DNA, wherein the tie strand (i) is attached to a nucleotide of the first double-stranded DNA molecule (1) by at least one covalent bond, and (ii) is attached to a nucleotide of the second double-stranded DNA molecule (2) by at least one covalent bond.

[0141] Furthermore, this document discloses methods and systems for forming multiple nucleic acid scaffolds. In some embodiments, the method includes: (a) providing a plurality of hairpin nucleic acids, wherein each hairpin nucleic acid is a sequential polynucleotide sequence, wherein each hairpin nucleic acid contains a barcode, and wherein at least two hairpin nucleic acids contain different barcodes; (b) attaching beads to a first end of each hairpin nucleic acid; and (c) attaching a second end of each hairpin nucleic acid to a bottom surface of a device; (d) decoding the barcode of each hairpin nucleic acid; (e) hybridizing a first spacer polynucleotide with each hairpin nucleic acid in a first spacer binding sequence of the plurality of hairpin nucleic acids, wherein the first spacer polynucleotide contains a polynucleotide sequence complementary to the first spacer binding sequence; and (f) hybridizing a second spacer polynucleotide with each hairpin nucleic acid in a second spacer binding sequence of the plurality of hairpin nucleic acids, wherein the second spacer polynucleotide contains a polynucleotide sequence complementary to the second spacer binding sequence. In some embodiments, a first spacer polynucleotide and a second spacer polynucleotide are provided together, wherein the first spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of a first spacer binding sequence of a plurality of hairpin nucleic acids, wherein the second spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of a second spacer binding sequence of a plurality of hairpin nucleic acids, and wherein the first spacer polynucleotide hybridizes with the first spacer binding sequence, and the second spacer polynucleotide hybridizes with the second spacer binding sequence.

[0142] In some embodiments, decoding of the barcode provides identification of the underlying nucleotide sequence of the hairpin nucleic acid 100. Therefore, in some embodiments, decoding allows identification of a first molecule-binding sequence 104 and a second molecule-binding sequence 105 of the hairpin nucleic acid, wherein a first candidate molecule and a second candidate molecule are anchored to the first molecule-binding sequence 104 and the second molecule-binding sequence 105, respectively. In some embodiments, at least one of the first molecule-binding sequence 104 and the second molecule-binding sequence 105 is a small hairpin nucleic acid. In some embodiments, both the first molecule-binding sequence 104 and the second molecule-binding sequence 105 are small hairpin nucleic acids.

[0143] To prepare the nucleic acid scaffold, in some embodiments, hairpin nucleic acids are linked to beads. Beads linked to the hairpin nucleic acid are injected into a flow cell to immobilize them to a feature portion of the flow cell. Immobilization is achieved by hybridizing a second adaptor sequence of the hairpin nucleic acid with a feature portion having a complementary nucleotide sequence. Feature portions having complementary polynucleotide sequences (SEQ ID NO:24) can be attached to the surface of the flow cell via covalent interactions (e.g., click chemistry between the azide-coated bottom surface of the flow cell and the polynucleotide sequence containing DBCO end groups). Unattached beads can be washed away.

[0144] Figure 11 A to Figure 11 C and Figure 22 A to Figure 22 C illustrates a graphical representation of a method for preparing a nucleic acid scaffold from hairpin nucleic acids located between a bead and a feature portion on the surface of the device. In short, as... Figure 11 As shown in A, hairpin nucleic acid 100 was then incubated with spacer polynucleotides to form holiday links. Subsequently, as... Figure 11 As shown in B, applying sufficient force to eliminate holiday connections leads to chain intrusion and the formation of a nucleic acid scaffold. Figure 11 As shown in C, during release, the binding sequences of the two spacer regions of the nucleic acid scaffold do not hybridize with each other. Or, as... Figure 22 As shown in Figure A, hairpin nucleic acid 100 was incubated together with spacer polynucleotides to form holiday links. Subsequently, as... Figure 22 As shown in B, applying sufficient force to eliminate holiday connections leads to chain intrusion and the formation of a nucleic acid scaffold. Figure 22 As shown in C, during release, the binding sequences of the two spacer regions of the nucleic acid scaffold do not hybridize with each other. Figure 23 It shows Figure 22 A to Figure 22 An exemplary trajectory of the event described in C. Hairpin nucleic acids containing a universal barcode sequence can be used to generate nucleic acid scaffolds by allowing chain intrusion. In some embodiments, one or more mutations present in the barcode sequence of the hairpin nucleic acid generated from the universal barcode sequence do not affect the chain intrusion efficiency of the spacer nucleotides.

[0145] Methods for determining binding interactions and the binding energy associated with binding interactions.

[0146] This document discloses methods and systems for determining binding interactions and binding energies related to binding interactions between at least two candidate molecules. In some embodiments, the at least two candidate molecules are selected proteins of interest, nucleic acids of interest, and small molecules of interest as described herein. In some embodiments, the at least two candidate molecules are proteins of interest. In some embodiments, the at least two candidate molecules are small molecules of interest. In some embodiments, the at least two candidate molecules are nucleic acids of interest. In some embodiments, the at least two candidate molecules are selected from both proteins of interest and nucleic acids of interest. In some embodiments, the at least two candidate molecules are selected from both proteins of interest and small molecules of interest. In some embodiments, the at least two candidate molecules are selected from both nucleic acids of interest and small molecules of interest.

[0147] This document discloses methods and systems for determining binding interactions between candidate molecules in real time. Furthermore, it discloses methods and systems for determining binding energies associated with binding interactions between candidate molecules. In some embodiments, the methods and systems are capable of detecting and analyzing molecular interactions between candidate molecules in real time. In some embodiments, the methods and systems include determining binding interactions between candidate molecules at the single-molecule level. In some embodiments, the methods and systems include determining at least two binding interactions between candidate molecules in parallel at the single-molecule level. In some embodiments, the methods and systems include using magnetic tweezers to determine binding interactions between candidate molecules. In some embodiments, the methods and systems include using optical tweezers to determine binding interactions between candidate molecules.

[0148] This document discloses a method and system for determining binding interactions (Kon and Koff) between a first candidate molecule and a second candidate molecule using a nucleic acid scaffold. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference amplitude of the nucleic acid scaffold in response to low-force (<0.01 pN) Brownian noise in the absence of the first and second candidate molecules; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby connecting the first candidate molecule and the second candidate molecule... Candidate molecules are attached to the first molecule binding sequence and the second molecule binding sequence, respectively, to form a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other; (f) by repeating (d), the amplitude of the Brownian noise of the screening nucleic acid scaffold in response to the same force applied in the absence of the first candidate molecule and the second candidate molecule is determined; and (g) interaction events between the two molecules are identified, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the molecule. A value corresponding to approximately 1 / 3 of the amplitude value indicates that there is a binding interaction between the first candidate molecule and the second candidate molecule under the applied force. Once these events are identified, in some embodiments, K is determined by considering all events that would have occurred within the time taken when the two molecules do not interact. on And K off This corresponds to the average time of all these detected events. In some embodiments, the first molecule binding sequence comprises a small hairpin nucleic acid, and the first gel sequence comprises a polynucleotide complementary to the small hairpin nucleic acid. In some embodiments, the second molecule binding sequence comprises a small hairpin nucleic acid, and the second gel sequence comprises a polynucleotide complementary to the small hairpin nucleic acid.

[0149] Furthermore, this paper discloses a method for using nucleic acid scaffolds to determine the binding interactions (K-interactions) between the first, second, and third candidate molecules. on and K offMethods and systems for [the study]. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference amplitude of the nucleic acid scaffold in response to low-force (<0.01 pN) Brownian noise in the absence of the first and second candidate molecules; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to [the gel sequence]. (f) The first and second molecule binding sequences are received, and a screening nucleic acid scaffold is formed, wherein the first and second candidate molecules are positioned along the screening nucleic acid scaffold such that the first and second candidate molecules are adjacent to or in contact with each other; (g) a third candidate molecule in solution is brought into contact with the screening nucleic acid scaffold; (h) the amplitude of the Brownian noise of the screening nucleic acid scaffold in the presence of the third candidate molecule is determined in response to the same low force applied in (d); and (f) an interaction event between the three molecules is identified, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the molecule. A value corresponding to approximately 1 / 3 of the amplitude value indicates the existence of binding interactions between the first, second, and third candidate molecules in solution under the applied force. Once these events are identified, Kon can be determined by considering all events that would take the time when two molecules do not interact, and Koff corresponds to the average time of all these detected events. In some embodiments, the first molecule binding sequence comprises a hairpin nucleic acid, and the first gel sequence comprises a polynucleotide complementary to the hairpin nucleic acid. In some implementations, the second molecule binding sequence comprises a hairpin nucleic acid, and the second gel sequence comprises a polynucleotide complementary to the hairpin nucleic acid.

[0150] Furthermore, this paper discloses a method for using nucleic acid scaffolds to determine the binding interactions (K-interactions) between the first, second, and third candidate molecules. on and K offMethods and systems for screening nucleic acid scaffolds. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are aligned along the screening scaffold. The nucleic acid scaffold is positioned such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other; (e) a reference amplitude of the Brownian noise of the nucleic acid scaffold in response to the force in the presence of the first candidate molecule and the second candidate molecule is determined; (f) a third candidate molecule in solution is brought into contact with the screening nucleic acid scaffold; (g) the amplitude of the Brownian noise of the screening nucleic acid scaffold in response to the same low force applied in (e) in the presence of the third candidate molecule is determined; (h) an interaction event between the three molecules is identified, wherein the amplitude of the Brownian noise decreases or remains unchanged compared to the reference amplitude under the same force in the presence of the first molecule and the second molecule. A decrease in Brownian noise in the presence of the third molecule indicates that the third molecule stabilizes the interaction between the first and second molecules. Conversely, if the amplitude of the Brownian noise remains unchanged in the presence of the third molecule, it indicates that the third molecule does not affect the interaction between the first and second molecules. In some embodiments, the first molecule binding sequence comprises a hairpin nucleic acid, and the first gel sequence comprises a polynucleotide complementary to the hairpin nucleic acid. In some implementations, the second molecule binding sequence comprises a hairpin nucleic acid, and the second gel sequence comprises a polynucleotide complementary to the hairpin nucleic acid.

[0151] This document discloses a method and system for determining the binding energy (enthalpy, entropy, and ΔG) between a first candidate molecule and a second candidate molecule using a nucleic acid scaffold. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference elongation length of the nucleic acid scaffold in response to a force in the absence of the first candidate molecule and the second candidate molecule; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby contacting the first candidate molecule with the first gel sequence. The first and second candidate molecules are respectively attached to the binding sequences of the first and second molecule molecules, forming a screening nucleic acid scaffold, wherein the first and second candidate molecules are positioned along the screening nucleic acid scaffold such that they are adjacent to or in contact with each other; (f) by repeating (d), the elongation length of the screening nucleic acid scaffold in response to the same force applied in the absence of the first and second candidate molecules is determined; (g) a difference is calculated, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and a reference elongation length. A non-zero difference indicates that there is a binding interaction between the first and second candidate molecules under the applied force. Conversely, a difference of zero indicates that there is no binding interaction between the first and second candidate molecules under the applied force. In some embodiments, the reference elongation length is determined in real time.

[0152] Furthermore, this document discloses a method and system for determining the binding energies (enthalpy, entropy, and ΔG) between a first candidate molecule, a second candidate molecule, and a third candidate molecule using a nucleic acid scaffold. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference elongation length of the nucleic acid scaffold in response to force in the absence of the first candidate molecule and the second candidate molecule; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein... The first and second candidate molecules are positioned along the screening nucleic acid scaffold such that the first and second candidate molecules are adjacent to or in contact with each other; (f) by repeating (d), the force required to achieve the same elongation length of the screening nucleic acid scaffold as a reference elongation length is determined (e.g., a force in the range of 0.01 pN to 10 pN); (g) a third candidate molecule in solution is brought into contact with the screening nucleic acid scaffold; (h) by repeating (d), the elongation length of the screening nucleic acid scaffold in the presence of the third candidate molecule is determined in response to the same force applied in (f); (I) This document discloses a method and system for determining the energy associated with the binding interaction between the first and second candidate molecules using a nucleic acid scaffold.In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence, and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a first force required to elongate the nucleic acid scaffold to a reference elongation length in the absence of the first candidate molecule and the second candidate molecule, wherein the reference elongation length is a length of the nucleic acid scaffold that increases independently of an increase in the amount of force applied to the nucleic acid scaffold, and wherein the first force is a minimum amount of force required to elongate the nucleic acid scaffold to the reference elongation length; (e) connecting the nucleic acid scaffold to the first gel sequence linked to the second candidate molecule. A first candidate molecule and a second candidate molecule linked to the second gel sequence are contacted, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, to form a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule can be adjacent to or in contact with each other; (f) by repeating (d), the force required to achieve an elongation length of the screening nucleic acid scaffold equal to the reference elongation length is determined; (g) a force difference is calculated, wherein the force difference is the difference between the force required to elongate the nucleic acid scaffold to the reference elongation length and the force required to elongate the screening nucleic acid scaffold to the reference elongation length. A non-zero force difference provides the binding energy associated with the binding interaction between the first candidate molecule and the second candidate molecule. Conversely, a difference of zero indicates that there is no binding interaction between the first candidate molecule and the second candidate molecule under the applied force. In some embodiments, the reference elongation length is determined in real time.

[0153] Furthermore, this document discloses methods and systems for determining binding energies related to binding interactions between a first candidate molecule, a second candidate molecule, and a third candidate molecule using a nucleic acid scaffold. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a first force required to elongate the nucleic acid scaffold to a reference elongation length in the absence of the first and second candidate molecules, wherein the reference elongation length is a length of the nucleic acid scaffold that increases independently of an increase in the amount of force applied to the nucleic acid scaffold, and wherein the first force is a minimum amount of force required to elongate the nucleic acid scaffold to the reference elongation length; (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a sieve. (f) Selecting a nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other; (d) determining, by repeating (d), a second force (e.g., a force in the range of 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) is required to achieve the same elongation length of the screening nucleic acid scaffold as the reference elongation length; (g) contacting the screening nucleic acid scaffold with a third candidate molecule in solution; (h) determining, by repeating (d), a third force (e.g., a force in the range of 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) is required to achieve the same elongation length of the screening nucleic acid scaffold as the reference elongation length in the presence of the third candidate molecule; (i) calculating a force difference, wherein the force difference is the difference between the first force and the second force, the second force and the third force, or the first force and the third force. A non-zero force difference between the first and second forces provides the binding energy associated with the binding interaction between the first and second candidate molecules. Conversely, a zero force difference between the first and second forces indicates that there is no binding interaction between the first and second candidates. Alternatively, a non-zero force difference between the second and third forces provides the binding energy associated with the binding interaction between the third candidate molecule and the first and second candidate molecules. Conversely, a zero force difference between the second and third forces indicates that there is no binding interaction between: (A) the third candidate molecule and the first candidate molecule, (B) the third candidate molecule and the second candidate molecule, or (C) the third candidate molecule, and the first and second candidate molecules.

[0154] In some implementations, the reference elongation length is determined by: (a) applying a force (e.g., in the range of 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) to the beads attached to the nucleic acid scaffold via a force application mechanism along an axis perpendicular to the bottom surface of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis; and (b) measuring the change in the position of the beads along the axis via a sensor to determine the reference elongation length of the nucleic acid scaffold.

[0155] In some implementations, the elongation length of the screening nucleic acid scaffold is determined by: (a) applying a force to beads attached to the nucleic acid scaffold along an axis perpendicular to the bottom surface of the device via a force application mechanism, the nucleic acid scaffold being attached to a first candidate molecule and a second candidate molecule, wherein the nucleic acid scaffold is configured to unfold in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis; and (b) determining a reference elongation length of the nucleic acid scaffold by measuring the change in the position of the beads along the axis via a sensor.

[0156] In some embodiments, the force applied to determine one or more of the reference elongation length and the elongation length of the screening nucleic acid scaffold includes at least 0.01 pN, at least 0.05 pN, at least 0.1 pN, at least 5 pN, at least 10 pN, at least 20 pN, at least 35 pN, at least 70 pN, or at least 95 pN. In some embodiments, the force does not exceed 100 pN. In some embodiments, the force is in the range of 0.01 pN to 10 pN, 0.01 pN to 8 pN, 0.01 pN to 5 pN, 0.01 pN to 3 pN, 0.05 pN to 10 pN, 0.05 pN to 8 pN, 0.05 pN to 5 pN, 0.05 pN to 3 pN, 0.1 pN to 10 pN, 0.1 pN to 8 pN, 0.1 pN to 5 pN, 0.1 pN to 3 pN, 2 pN to 10 pN, 2 pN to 7 pN, 2 pN to 5 pN, or 5 pN to 10 pN. In some embodiments, the force is less than 10 pN. In some embodiments, the force is 10 pN. In some embodiments, the force is less than 5 pN. In some embodiments, the force is 5 pN. In some embodiments, the force is greater than 0.1 pN. In some implementations, the force is greater than 0.01 pN.

[0157] Figure 13 A to Figure 13 B illustrates a graphical representation of the method for determining the binding interaction between two candidate molecules and the energy associated with the binding interaction using the system described herein. Figure 13As shown in Figure A, the system includes a nucleic acid screening scaffold comprising a nucleic acid scaffold and two candidate molecules anchored to the scaffold via two gel sequences, wherein the two candidate molecules interact with each other. Figure 13 As shown in Figure B, under the applied force, there is a binding interaction between the two candidate molecules, and the stretching of the nucleic acid scaffold is not as great as the reference elongation length of the nucleic acid scaffold. When... Figure 13 As shown in Figure C, when the interaction between the two candidate molecules is disrupted under the applied force, the nucleic acid scaffold stretches to a length similar to the reference elongation length observed before the interaction (not shown). Binding interactions and the binding energy of the interaction between the two candidate molecules due to the reduction in displacement in the Z-axis can be monitored by cyclically applying forces between low forces (e.g., about 0.1 pN or less, or about 0.01 pN) and high forces (e.g., 0.1 pN to 25 pN, depending on the strength of the interaction). The binding interaction can be disrupted by applying a force in a direction perpendicular to the surface of the device (e.g., the Z-direction). Once the interaction is disrupted, the screening nucleic acid scaffold is fully stretched and causes a Z-axis jump corresponding to the size of the nucleic acid scaffold. In some embodiments, the presence of binding interactions between the two candidate molecules corresponds to low Brownian noise, relative to the high Brownian noise observed when there is no binding interaction between the two candidate molecules.

[0158] This paper also describes a method for determining the binding interaction between two candidate molecules and the energy associated with the binding interaction using the system described herein, wherein at least one molecular binding sequence of the hairpin nucleic acid in the system contains a small hairpin nucleic acid ( Figure 20A The exemplary first-molecule binding sequences of the hairpin nucleic acids described herein include SEQ ID NO: 50 and 51 (…). Figure 20B and Figure 20C The exemplary second-molecule binding sequence of the hairpin nucleic acid described herein includes SEQ ID NO: 52. Figure 20D The original traces of the binding between the gel sequence attached to the candidate molecule and the molecular binding sequence of the hairpin nucleic acid described herein are shown. Figure 20A and Figure 20D As shown, the binding of one gel sequence to the corresponding complementary molecular binding sequence of the hairpin nucleic acid results in the loss of one hop from the scaffold molecule, and the binding of two gel sequences to the corresponding complementary molecular binding sequences of the hairpin nucleic acid results in the loss of two hops. Figure 20A and Figure 20D The analysis also showed that with increasing force (e.g., 5 pN, 10 pN or more), holiday linkages were eliminated and hairpins were converted into dsDNA. This resulted in the disappearance of small jumps due to the presence of small hairpin sequences.

[0159] In some embodiments, the methods described herein advantageously determine the binding interaction between two or more candidate molecules under an applied force in the range of 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN. In some embodiments, the force is magnetic, and the beads are magnetic beads.

[0160] Methods for determining the dynamics of the combination

[0161] This document discloses methods and systems for determining the binding kinetics between at least two candidate molecules. In some embodiments, the at least two candidate molecules are a selected protein of interest, a nucleic acid of interest, and a small molecule of interest as described herein. In some embodiments, the at least two candidate molecules are proteins of interest. In some embodiments, the at least two candidate molecules are small molecules of interest. In some embodiments, the at least two candidate molecules are nucleic acids of interest. In some embodiments, the at least two candidate molecules are selected from both proteins of interest and nucleic acids of interest. In some embodiments, the at least two candidate molecules are selected from both proteins of interest and small molecules of interest. In some embodiments, the at least two candidate molecules are selected from both nucleic acids of interest and small molecules of interest.

[0162] This document discloses methods and systems for determining the binding dynamics of candidate molecules in real time. In some embodiments, the methods and systems are capable of detecting binding interactions between candidate molecules in real time and analyzing molecular interactions to determine binding dynamics and energy. In some embodiments, the methods and systems include determining binding dynamics and energy at the single-molecule level. In some embodiments, the methods and systems include determining at least two binding dynamics or energy between candidate molecules in parallel at the single-molecule level. In some embodiments, the methods and systems include using magnetic tweezers to determine binding dynamics.

[0163] This document discloses methods and systems for determining the binding kinetics and energy of binding interactions between a first candidate molecule and a second candidate molecule. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference elongation length of the nucleic acid scaffold in response to a force in the absence of the first candidate molecule and the second candidate molecule; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule... The first and second candidate molecules are positioned along the screening nucleic acid scaffold such that the first and second candidate molecules are adjacent to or in contact with each other; (f) by repeating (d), the elongation length of the screening nucleic acid scaffold in response to the same force applied in the absence of the first and second candidate molecules is determined; (g) a difference is calculated, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and a reference elongation length; (h) after (g), the force applied to the beads by the force application mechanism is removed, resulting in relaxation of the screening nucleic acid scaffold; and (i) (f) to (h) are repeated and the difference between the elongation length of the screening nucleic acid scaffold attached to the first and second candidate molecules and the reference elongation length as a function of time is calculated. In some embodiments, the method herein further includes: (i) determining the K of the binding of the second molecule to the first candidate molecule. on K on The calculation is based on the number of repetitions (f) to (h) that result in the difference between the elongation length of the screening nucleic acid scaffold and a reference elongation length. In some implementations, the K of the interaction is determined based on the number of cycles observed to have blockage. on In some embodiments, the method described herein further includes: (h) determining the KB of the binding of the second molecule to the first candidate molecule. off K off The K value is calculated based on the time length during which the elongation of the screening nucleic acid scaffold differs from the reference elongation during each cycle of repetitions (f) through (h). In some implementations, the K value of the interaction is determined based on the duration of the blockage. offIn some embodiments, the first molecular binding sequence of the nucleic acid scaffold described herein comprises a hairpin nucleic acid, wherein the first gel sequence as described herein comprises a polynucleotide complementary to the hairpin nucleic acid. In some embodiments, the second molecular binding sequence of the nucleic acid scaffold described herein comprises a hairpin nucleic acid, wherein the second gel sequence as described herein comprises a polynucleotide complementary to the hairpin nucleic acid.

[0164] Furthermore, this document discloses a method and system for determining binding kinetics of binding interactions between a first candidate molecule, a second candidate molecule, and a third candidate molecule using a nucleic acid scaffold. In some embodiments, the method includes: (a) providing a nucleic acid scaffold as described herein; (b) providing an apparatus as described herein; (c) providing a first candidate molecule as described herein linked to a first gel sequence and a second candidate molecule as described herein linked to a second gel sequence; (d) determining a reference elongation length of the nucleic acid scaffold in response to a force in the absence of the first candidate molecule and the second candidate molecule; and (e) contacting the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first... The candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are in contact with each other; (f) by repeating (d), the force required to achieve the same screening nucleic acid scaffold elongation length as the reference elongation length is determined (e.g., a force in the range of 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) is determined; (g) the third candidate molecule is brought into contact with the screening nucleic acid scaffold; (h) by repeating (d), the elongation length of the screening nucleic acid scaffold in the presence of the third candidate molecule is determined in response to the same force applied in (f); (i) the difference is calculated, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and the reference elongation length. In some embodiments, the method further includes: (j) after (i), removing the force applied to the beads by the force application mechanism, thereby causing relaxation of the test screening nucleic acid scaffold; and (k) repeating (e) to (j) and calculating, as a function of time, the difference in the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule. In some embodiments, the method further includes: (j) determining the K-value of the binding of the third candidate molecule to the first molecule and the second molecule. on , where K onThe calculation is based on the number of repetitions (h) to (j) of the number of cycles that result in the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule. In some embodiments, the method further includes: (j) determining the K-value of the binding of the third candidate molecule to the first candidate molecule and the second candidate molecule. off , where K off The calculation is based on the average time length of the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule during each cycle of repetitions (h) to (j). In some embodiments, the first molecule-binding sequence of the nucleic acid scaffold described herein comprises a small hairpin nucleic acid, wherein the first gel sequence as described herein comprises a polynucleotide complementary to the small hairpin nucleic acid. In some embodiments, the second molecule-binding sequence of the nucleic acid scaffold described herein comprises a small hairpin nucleic acid, wherein the second gel sequence as described herein comprises a polynucleotide complementary to the small hairpin nucleic acid.

[0165] Figures 15A to 15B An exemplary trace is shown for determining the binding dynamics between two candidate molecules. Figures 15A to 15B As shown, the presence of binding interactions between two candidate molecules corresponds to low Brownian noise, compared to the high Brownian noise observed when there is no binding interaction between them. Similarly, Figure 16 Exemplary changes in the Brownian motion of beads anchored to nucleic acid scaffolds are shown in the presence and absence of two candidate molecules, ACE2 and SARS-CoV2 RBD. Figure 26A An exemplary trace is shown for determining the binding dynamics between two candidate molecules, ACE2 and RBD. Figure 26B As shown, the presence of a binding interaction between ACE2 and RBD corresponds to low Brownian noise, compared to the high Brownian noise observed in the absence of binding interactions. In some embodiments, the force is kept constant throughout the experiment. Therefore, the constant applied force can range from almost 0 pN to high forces (e.g., 25 pN, depending on the strength of the interaction). In some embodiments, the force is maintained for at least 5 minutes, or at least 10 minutes, or at least 20 minutes, or at least 40 minutes, or at least 60 minutes, or at least 120 minutes. In some embodiments, the method further includes determining the K-value of the binding between the first and second molecules. on K on The calculation is based on the number of times low Brownian noise is observed during the total duration of the experiment. In some embodiments, the method further includes determining the K-value for the binding of the first candidate molecule and the second candidate molecule. off K off It is calculated based on the average time length of the observed low Brownian noise.

[0166] Filtering methods

[0167] This document discloses methods and systems for screening candidate molecules. In some embodiments, candidate molecules are selected proteins of interest, nucleic acids of interest, small molecules of interest, or combinations thereof, as described herein.

[0168] This document discloses methods and systems for real-time screening of candidate molecules. In some embodiments, the methods and systems are capable of determining the binding strength interactions between candidate molecules in real time, analyzing the molecular interactions of each candidate molecule, determining the binding kinetics of each candidate molecule, determining the energy of the interactions, and thus comparing the candidate molecules with each other for screening. In some embodiments, the methods and systems include using magnetic tweezers to screen candidate molecules.

[0169] This article discloses a method and system for determining binding interactions between a first candidate molecule and a plurality of second candidate molecules using the nucleic acid scaffold described herein. In some embodiments, the method includes: (a) providing an apparatus as described herein, wherein the apparatus includes a plurality of nucleic acid scaffolds, each nucleic acid scaffold positioned along an axis perpendicular to a bottom surface of a chamber of the apparatus, and each of the plurality of nucleic acid scaffolds being connected at one end to a bead and at the other end to a feature of the bottom surface of the apparatus; (b) determining a reference elongation length of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules by: (i) applying a force (e.g., 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) to the bead attached to each of the plurality of nucleic acid scaffolds along the axis perpendicular to the bottom surface of the apparatus via a force application mechanism, wherein each of the plurality of nucleic acid scaffolds is configured to unfold in response to the applied force, thereby causing a change in the position of the bead attached to each of the plurality of nucleic acid scaffolds along the axis; and (ii) measuring the position of the bead along the axis via a sensor. (c) To determine a reference elongation length for each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules; (d) To contact the plurality of nucleic acid scaffolds with the first candidate molecule, thereby anchoring the first candidate molecule to each of the plurality of nucleic acid scaffolds; (e) To contact the plurality of nucleic acid scaffolds with the plurality of second candidate molecules, thereby anchoring one of the second candidate molecules to each of the plurality of nucleic acid scaffolds, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule and the second candidate molecule of each of the plurality of screening nucleic acid scaffolds are positioned such that the first candidate molecule and the second candidate molecule are in contact with each other; (f) To determine the elongation length of each of the plurality of screening nucleic acid scaffolds in response to a force (e.g., 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN) by repeating (d); and (c) To calculate the difference between the reference elongation length and the elongation length of each of the plurality of screening nucleic acid scaffolds. A non-zero difference indicates that a binding interaction exists between the first and second candidate molecules under the applied force. Conversely, a difference of zero indicates that no binding interaction exists between the first and second candidate molecules under the applied force. In some embodiments, the reference elongation length is determined in real time.

[0170] Furthermore, this document discloses a method and system for determining binding interactions between a first candidate molecule, a second candidate molecule, and a plurality of third candidate molecules using the nucleic acid scaffolds described herein. In some embodiments, the method includes: (a) providing an apparatus as described herein, wherein the apparatus includes a plurality of nucleic acid scaffolds, each nucleic acid scaffold positioned along an axis perpendicular to the bottom surface of a chamber of the apparatus, and each of the plurality of nucleic acid scaffolds being connected at one end to a bead and at the other end to a feature portion of the bottom surface of the apparatus; (b) determining a reference elongation length of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the second candidate molecule by: (i) applying a force along the axis perpendicular to the bottom surface of the apparatus via the force application mechanism. (e.g., 0.01pN to 10pN, 0.05pN to 10pN, or 0.1pN to 10pN) is applied to the bead attached to each of the plurality of nucleic acid scaffolds, wherein each of the plurality of nucleic acid scaffolds is configured to unfold in response to the applied force, thereby causing a change in the position of the bead attached to each of the plurality of nucleic acid scaffolds along the axis, and (ii) the change in the position of the bead along the axis is measured via a sensor to determine the position of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules. (c) Contact the plurality of nucleic acid scaffolds with the first candidate molecule, thereby anchoring the first candidate molecule to each of the plurality of nucleic acid scaffolds; (d) Contact the plurality of nucleic acid scaffolds with the plurality of second candidate molecules, thereby anchoring the second candidate molecule to each of the plurality of nucleic acid scaffolds, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule and the second candidate molecule of each of the plurality of screening nucleic acid scaffolds are positioned such that the first candidate molecule and the second candidate molecule are in contact with each other; (e) Determine the achievement of the reference elongation length by repeating (d). (b) applying the same force required for the same length of elongation of the screening nucleic acid scaffold (e.g., 0.01 pN to 10 pN, 0.05 pN to 10 pN, or 0.1 pN to 10 pN); (f) contacting the plurality of third candidate molecules with the plurality of screening nucleic acid scaffolds; and (g) by repeating (b), determining the length of elongation of each of the plurality of third candidate molecules in response to the same force applied in (e) in the presence and absence of at least one of the plurality of third candidate molecules; and (g) calculating a difference, wherein the difference is the difference between the length of elongation of the screening nucleic acid scaffold and a reference length of elongation. If there is no measurable difference between the length of elongation of the screening nucleic acid scaffold and the reference length of elongation (“zero difference”), no binding interaction is detected.If there is a measurable difference (“non-zero difference”) between the elongation length of the screening nucleic acid scaffold and the reference elongation length, this indicates that there is a binding interaction between the third candidate molecule and the first and second candidate molecules under the applied force. Conversely, a zero difference indicates that there is no binding interaction between: (A) the third candidate molecule and the first candidate molecule, (B) the third candidate molecule and the second candidate molecule, or (C) the third candidate molecule, with the first and second candidate molecules.

[0171] In some embodiments, each of the plurality of nucleic acid scaffolds described herein contains a barcode sequence. In some embodiments, at least two of the plurality of nucleic acid scaffolds contain barcode sequences that are different from each other. In some embodiments, at least two of the plurality of nucleic acid scaffolds contain barcode sequences located at different positions relative to each other. In some embodiments, the method described herein further includes determining the identity of hairpin nucleic acids based on barcodes. In some embodiments, the identity of hairpin nucleic acids is determined by detecting the position of one or more modified nucleotides in the barcode. In some embodiments, the method described herein further includes preparing nucleic acid scaffolds using any of the methods described herein. For example, in some embodiments, the method herein further includes: (a) providing a plurality of hairpin nucleic acids, wherein each of the plurality of hairpin nucleic acids is a continuous polynucleotide sequence, wherein each of the plurality of hairpin nucleic acids contains a barcode, wherein at least two of the plurality of hairpin nucleic acids contain different barcodes; (b) attaching beads to a first end of each of the plurality of hairpin nucleic acids; and (c) attaching a second end of each of the plurality of hairpin nucleic acids to a bottom surface of the device; (d) decoding the barcode of each of the plurality of hairpin nucleic acids; (e) hybridizing a first spacer polynucleotide with each of the plurality of hairpin nucleic acids in a first spacer binding sequence, wherein the first spacer polynucleotide contains a polynucleotide sequence complementary to the first spacer binding sequence; and (f) hybridizing a second spacer polynucleotide with each of the plurality of hairpin nucleic acids in a second spacer binding sequence, wherein the second spacer polynucleotide contains a polynucleotide sequence complementary to the second spacer binding sequence. In some embodiments, a first spacer polynucleotide and a second spacer polynucleotide are provided together, wherein the first spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of a first spacer binding sequence of a plurality of hairpin nucleic acids, wherein the second spacer polynucleotide comprises a polynucleotide sequence complementary to at least a portion of a second spacer binding sequence of a plurality of hairpin nucleic acids, and wherein the first spacer polynucleotide hybridizes with the first spacer binding sequence, and the second spacer polynucleotide hybridizes with the second spacer binding sequence.

[0172] In some embodiments, the method described herein also includes testing the activity of the identified candidate molecule as a medicament for the prevention or treatment of diseases directly or indirectly related to the candidate molecule.

[0173] In some embodiments, attaching multiple hairpin nucleic acids to multiple feature portions includes covalently attaching a second adaptor 107 of each of the multiple hairpin nucleic acids to one of the multiple feature portions on the bottom surface of the device. In some embodiments, attaching multiple hairpin nucleic acids to multiple feature portions includes non-covalently attaching the second adaptor 107 of each of the multiple hairpin nucleic acids to one of the multiple feature portions on the bottom surface of the device. In some embodiments, attaching multiple hairpin nucleic acids to multiple feature portions includes attaching the second adaptor 107 of each of the multiple hairpin nucleic acids to one or more feature portions on the bottom surface of the device by hybridization, wherein the second adaptor 107 comprises a polynucleotide sequence complementary to the polynucleotide sequence of the feature portion. In some embodiments, each feature portion of the chamber comprises a single nucleic acid scaffold among multiple nucleic acid scaffolds.

[0174] In some implementations, the change in elongation length of each of a variety of screened nucleic acids is measured in real time by using a magnet to apply force (advantageously a series of constant forces or a series of incremental forces) to nucleic acid molecules and by measuring the movement of beads attached to the nucleic acid.

[0175] Exemplary Implementation

[0176] Implementation Scheme 1: A nucleic acid scaffold for determining the binding interaction between a first candidate molecule and a second candidate molecule, wherein the nucleic acid scaffold comprises:

[0177] (a) A continuous polynucleotide sequence comprising:

[0178] (i) Attached to the first end of the bead, wherein the first end contains a first molecular binding sequence.

[0179] (ii) A second end attached to the bottom surface of the device, wherein the second end contains a second molecular binding sequence, and

[0180] (iii) The intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises:

[0181] (I) The first lead containing the barcode forms a sequence.

[0182] (II) A second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence hybridizes with the second lead-forming sequence, and

[0183] (III) A loop connecting the first lead-forming sequence and the second lead-forming sequence;

[0184] (b) a first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and

[0185] (c) A second spacer polynucleotide having a polynucleotide sequence complementary to the second lead-forming sequence.

[0186] The first spacer polynucleotide and the second spacer polynucleotide hybridize with the middle portion, and

[0187] The nucleic acid scaffold requires a force of 0.01pN to 10pN, 0.05pN to 10pN, or 0.1pN to 10pN to be applied to the bead along an axis perpendicular to the bottom surface of the device to unfold the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold.

[0188] Implementation Scheme 2. The nucleic acid scaffold according to Implementation Scheme 1, wherein the barcode sequence comprises one or more modified nucleotides.

[0189] Implementation Scheme 3. The nucleic acid scaffold according to any one of Implementation Schemes 1 to 2, wherein the intermediate portion further comprises a first linker sequence and a second linker sequence, wherein the first linker sequence is located between the first molecule binding sequence and the first lead-forming sequence, and wherein the second linker sequence is located between the second molecule binding sequence and the second lead-forming sequence, and wherein the first linker sequence and the second linker sequence are not complementary to each other.

[0190] Implementation Scheme 4. The nucleic acid scaffold according to any one of Implementation Schemes 1 to 3, wherein the first spacer polynucleotide and the second spacer polynucleotide are connected to each other by a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop.

[0191] Implementation Scheme 5. The nucleic acid scaffold according to any one of Implementation Schemes 1 to 4, wherein the first molecular binding sequence and the second molecular binding sequence each independently comprise a hairpin nucleic acid having a size in the range of 5 to 100 bases.

[0192] Implementation Scheme 6. A nucleic acid screening scaffold, the nucleic acid screening scaffold comprising:

[0193] (a) A nucleic acid scaffold according to any one of implementation schemes 1 to 5;

[0194] (b) ligating a first candidate molecule to a first gel sequence, wherein the first gel sequence hybridizes with the first molecule-binding sequence; and

[0195] (c) A second candidate molecule is attached to the second gel sequence, wherein the second gel sequence hybridizes with the second molecule binding sequence.

[0196] Implementation Scheme 7. An apparatus comprising:

[0197] (a) A chamber disposed within the device, wherein the chamber includes a bottom surface;

[0198] (b) a force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; and

[0199] (c) A nucleic acid scaffold according to any one of embodiments 1 to 5 or a screening nucleic acid scaffold according to embodiment 6.

[0200] Implementation Scheme 8. A method for determining the binding interaction between a first candidate molecule and a second candidate molecule, the method comprising:

[0201] (a) Providing a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device;

[0202] (b) A device is provided, the device comprising:

[0203] (i) a chamber disposed within the device, wherein the chamber includes a bottom surface capable of incorporating an anchoring sequence of any molecule according to embodiments 1 to 5, and

[0204] (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold;

[0205] (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule and wherein the second gel sequence is complementary to the binding sequence of the second molecule;

[0206] (d) Determine the reference elongation length of the nucleic acid scaffold in response to force in the absence of the first and second candidate molecules in real time by the following method:

[0207] (i) A force of 0.01 pN to 50 pN is applied to the bead attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the bead attached to the nucleic acid scaffold along the axis, and

[0208] (ii) The change in the position of the bead along the axis is measured by a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule;

[0209] (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to each other;

[0210] (f) By repeating (d), determine the elongation length of the screening nucleic acid scaffold in response to the same force applied in the absence of the first candidate molecule and the second candidate molecule; and

[0211] (g) Calculate the difference, where the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and the reference elongation length.

[0212] A non-zero difference indicates that the binding interaction exists between the first candidate molecule and the second molecule under the applied force, and a zero difference indicates that the binding interaction does not exist between the first candidate molecule and the second candidate molecule under the applied force.

[0213] Implementation Scheme 9. The method according to Implementation Scheme 8, further comprising determining the binding kinetics of the binding interaction between the first candidate molecule and the second candidate molecule by: (h) after (g), removing the force applied to the bead by the force application mechanism, thereby causing relaxation of the screening nucleic acid scaffold; and (i) repeating (f) to (h) and calculating the difference between the elongation length of the screening nucleic acid scaffold as a function of time and the reference elongation length.

[0214] Implementation Scheme 10. The method according to Implementation Scheme 9, further comprising: (i) determining the K-value for the binding of the second molecule to the first candidate molecule. on , where K on The calculation is based on the number of cycles (f) to (h) that result in the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length.

[0215] Implementation Scheme 11. The method according to Implementation Scheme 9, further comprising: (h) determining the K-force of the binding of the second molecule to the first candidate molecule under a predetermined force. off , where K offThe calculation is based on the length of time during which the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length exists in each cycle of repetition (f) to (h) under the limiting force.

[0216] Implementation Scheme 12. A method for determining binding interactions among a first candidate molecule, a second candidate molecule, and a third candidate molecule, the method comprising:

[0217] (a) Providing a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device;

[0218] (b) A device is provided, the device comprising:

[0219] (i) a chamber disposed within the device, wherein the chamber includes a bottom surface capable of incorporating the anchoring sequence 107 of any molecule as described in embodiments 1 to 5, and

[0220] (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold;

[0221] (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule and wherein the second gel sequence is complementary to the binding sequence of the second molecule;

[0222] (d) Determine the reference elongation length of the nucleic acid scaffold in the absence of the first and second candidate molecules by the following method:

[0223] (i) A force of 0.01 pN to 50 pN is applied to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and

[0224] (ii) The change in the position of the bead along the axis is measured by a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule;

[0225] (e) The nucleic acid scaffold is brought into contact with the first candidate molecule and the second candidate molecule, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to each other;

[0226] (f) By repeating (d), determine the force required to achieve the same elongation length as the reference elongation length for the screening nucleic acid scaffold, wherein the required force is in the range of 0.01 pN to 50 pN;

[0227] (g) bringing the third candidate molecule into contact with the screening nucleic acid scaffold; and

[0228] (h) By repeating (d), determine the elongation length of the screening nucleic acid scaffold in the presence of the third candidate molecule and in response to the same force applied in (f); and

[0229] (i) Calculate the difference, where the difference is the length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule.

[0230] A non-zero difference indicates that a binding interaction exists between the third candidate molecule and the first and second candidate molecules under the applied force, and a zero difference indicates that no such binding interaction exists between them:

[0231] (A) The third candidate molecule and the first candidate molecule,

[0232] (B) The third candidate molecule and the second candidate molecule, or

[0233] (C) The third candidate molecule under the applied force, together with the first candidate molecule and the second candidate molecule.

[0234] Implementation Scheme 13. The method according to Implementation Scheme 12, further comprising determining the binding kinetics of the binding interaction between the first candidate molecule, the second candidate molecule and the third candidate molecule by: (j) after (i), removing the force applied to the bead by the force application mechanism, thereby causing relaxation of the test screening nucleic acid scaffold; and (k) repeating (e) to (j) and calculating the difference in the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule as a function of time.

[0235] Implementation Scheme 14. The method according to Implementation Scheme 12, further comprising: (j) determining the K-value of the binding of the third candidate molecule to the first molecule and the second molecule. on , where K on The calculation is based on the number of cycles (h) to (j) that result in the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule.

[0236] Implementation Scheme 15. The method according to Implementation Scheme 12, further comprising: (j) determining the K-force of the binding of the third candidate molecule to the first candidate molecule and the second candidate molecule under a predetermined force. off , where K off The calculation is based on the length of time during which the elongation of the screening nucleic acid scaffold differs between the presence and absence of the third candidate molecule during each cycle of repetitions (h) to (j).

[0237] Implementation Scheme 16. A method for screening binding interactions between a first candidate molecule and a plurality of second candidate molecules, the method comprising:

[0238] (a) A device is provided, the device comprising:

[0239] (i) A chamber disposed within the device, wherein the chamber includes a bottom surface.

[0240] (ii) Force-applying mechanism, and

[0241] (iii) A plurality of nucleic acid scaffolds positioned along an axis perpendicular to the bottom surface of the chamber of the device, wherein each of the plurality of nucleic acid scaffolds includes a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein each of the plurality of nucleic acid scaffolds is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the device;

[0242] (b) Determine the reference elongation length of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules by the following method:

[0243] (i) A force of 0.01 pN to 50 pN is applied via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device to the bead attached to each of the plurality of nucleic acid scaffolds, wherein each of the plurality of nucleic acid scaffolds is configured to deploy in response to the applied force, thereby causing a change in the position of the bead attached to each of the plurality of nucleic acid scaffolds along the axis, and

[0244] (ii) The change in the position of the bead along the axis is measured by a sensor to determine a reference elongation length for each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules;

[0245] (c) Contact the plurality of nucleic acid scaffolds with the first candidate molecule linked to the first gel sequence, thereby anchoring the first candidate molecule to each of the plurality of nucleic acid scaffolds, wherein the first gel sequence is complementary to the first molecule binding sequence;

[0246] (d) Contacting the plurality of nucleic acid scaffolds with the plurality of second candidate molecules each attached to the second gel sequence, thereby anchoring one of the second candidate molecules to each of the plurality of nucleic acid scaffolds, wherein the second gel sequence is complementary to the second molecule binding sequence, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule and the second candidate molecule of each of the plurality of screening nucleic acid scaffolds are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other;

[0247] (e) By repeating (d), the elongation length of each of the plurality of screening nucleic acid scaffolds in response to forces ranging from 0.01 pN to 50 pN is determined; and

[0248] (f) Calculate the difference between the reference elongation length and the elongation length of each of the plurality of screening nucleic acid scaffolds.

[0249] The difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length indicates that the first candidate molecule and the second candidate molecule anchored to the screening nucleic acid have a binding interaction with each other under the applied force, and the absence of the difference indicates that there is no binding interaction between the first candidate molecule and the second candidate molecule under the applied force.

[0250] Implementation Scheme 17. The method according to Implementation Scheme 16, wherein at least two nucleic acid scaffolds in the nucleic acid scaffold contain barcode sequences located at different positions relative to each other.

[0251] Implementation Scheme 18. The method according to Implementation Scheme 16, wherein at least two nucleic acid scaffolds in the nucleic acid scaffold contain barcode sequences that are different from each other.

[0252] Implementation Scheme 19. The method according to Implementation Scheme 17 or 18, further comprising determining the identity of the hairpin nucleic acid based on the barcode prior to (b).

[0253] Implementation Scheme 20. The method according to Implementation Scheme 19, wherein the identity of the hairpin nucleic acid is determined by detecting the position of one or more modified nucleotides in the barcode.

[0254] Implementation Scheme 21. The method according to Implementation Scheme 20, further comprising eliminating holiday links, wherein the holiday links are formed by hybridizing the first linker sequence and the second sequence to form the nucleic acid scaffold via the first linker sequence binding region of the first spacer polynucleotide and the second linker sequence binding region of the second spacer polynucleotide, respectively.

[0255] Implementation Scheme 22. A method for screening binding interactions between a first candidate molecule, a second candidate molecule, and a plurality of third candidate molecules, the method comprising:

[0256] (a) A device is provided, the device comprising:

[0257] (i) A chamber disposed within the device, wherein the chamber includes a bottom surface.

[0258] (ii) a force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold, and

[0259] (iii) A nucleic acid scaffold positioned along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold includes a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein the nucleic acid scaffold is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the device;

[0260] (b) Determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule by the following method:

[0261] (i) A force of 0.01 pN to 50 pN is applied to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and

[0262] (ii) The change in the position of the bead along the axis is measured by a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule;

[0263] (c) Contact the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence, thereby anchoring the first candidate molecule to the nucleic acid scaffold;

[0264] (d) Contact the nucleic acid scaffold with the second candidate molecule linked to the second gel sequence to anchor the second candidate molecule to the nucleic acid scaffold, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule of the screening nucleic acid scaffold are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other.

[0265] (e) By repeating (d), determine the force required to achieve the same elongation length as the reference elongation length for the screening nucleic acid scaffold, wherein the force is in the range of 0.01 pN to 50 pN;

[0266] (f) Contact the multiple third candidate molecules with the screening nucleic acid scaffold;

[0267] (g) By repeating (b), determine the elongation length of the screening nucleic acid scaffold in response to the same force applied in (e) in the presence of at least one of the plurality of third candidate molecules; and

[0268] (h) Calculate the difference between the reference elongation length and the elongation length of the screening nucleic acid scaffold.

[0269] A non-zero difference indicates that a binding interaction exists between the third candidate molecule and the first and second candidate molecules under the applied force, and a zero difference indicates that no such binding interaction exists between them:

[0270] (A) The third candidate molecule and the first candidate molecule,

[0271] (B) The third candidate molecule and the second candidate molecule, or

[0272] (C) The third candidate molecule under the applied force, together with the first candidate molecule and the second candidate molecule.

[0273] Implementation Scheme 23: A method for determining the binding interaction between a first candidate molecule and a second candidate molecule, the method comprising:

[0274] (a) Providing a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device;

[0275] (b) A device is provided, the device comprising:

[0276] (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and

[0277] (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold;

[0278] (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule and wherein the second gel sequence is complementary to the binding sequence of the second molecule;

[0279] (d) Determine the amplitude of the Brownian noise of the nucleic acid scaffold in response to a force of less than 0.01 pN in the absence of the first candidate molecule and the second candidate molecule;

[0280] (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, and thereby the first candidate molecule and the second candidate molecule are respectively attached to the first molecule binding sequence and the second molecule binding sequence, and a screening nucleic acid scaffold is formed, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other;

[0281] (f) By repeating (d), determine the amplitude of the Brownian noise of the screening nucleic acid scaffold in response to the same force used in the absence of the first and second candidate molecules; and

[0282] (g) Identify the interaction event between the two molecules, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the molecule.

[0283] Implementation Scheme 24: A method for determining binding interactions among a first candidate molecule, a second candidate molecule, and a third candidate molecule, the method comprising:

[0284] (a) Providing a nucleic acid scaffold according to any one of embodiments 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device;

[0285] (b) A device is provided, the device comprising:

[0286] (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and

[0287] (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold;

[0288] (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule and wherein the second gel sequence is complementary to the binding sequence of the second molecule;

[0289] (d) Determine the reference amplitude of the Brownian noise of the nucleic acid scaffold in response to a force of less than 0.01 pN in the absence of the first candidate molecule and the second candidate molecule;

[0290] (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, and thereby the first candidate molecule and the second candidate molecule are respectively attached to the first molecule binding sequence and the second molecule binding sequence, and a screening nucleic acid scaffold is formed, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other;

[0291] (f) Contact the third candidate molecule in the solution with the screening nucleic acid scaffold;

[0292] (g) Determine the amplitude of the Brownian noise of the screening nucleic acid scaffold in response to the same force applied in (d) in the presence of the third candidate molecule; and

[0293] (h) Identify the interaction event between the three molecules, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the three molecules.

[0294] Implementation Scheme 25. A kit for determining the binding interaction between a first candidate molecule and a second candidate molecule, the kit comprising:

[0295] (a) Nucleic acid, which contains:

[0296] (i) a continuous polynucleotide sequence comprising:

[0297] (I) A first end, comprising: (A) a first linker for attachment to a bead, and (B) a first molecule binding sequence;

[0298] (II) A second end comprising: (A) a second adaptor for attaching the nucleic acid to the bottom surface of the device, and (B) a second molecule binding sequence;

[0299] (III) The intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises:

[0300] 1. The first lead containing the barcode forms a sequence.

[0301] 2. A second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence hybridizes with the second lead-forming sequence, and

[0302] 3. A loop connecting the first lead-forming sequence and the second lead-forming sequence;

[0303] (ii) a first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and

[0304] (iii) A second spacer polynucleotide having a polynucleotide sequence complementary to the second lead-forming sequence.

[0305] When the nucleic acid is attached to the bottom surface of the device as a nucleic acid scaffold, a force of 10 pN to 30 pN is required to unfold the hairpin nucleic acid along an axis perpendicular to the bottom surface of the device; and

[0306] (b) A bead containing an anchoring molecule configured to bind to the first adaptor at the first end of the continuous polynucleotide sequence of the nucleic acid.

[0307] Implementation Scheme 26. The kit according to Implementation Scheme 25, wherein the first molecule binding sequence and the second molecule binding sequence each independently comprise a small hairpin nucleic acid having a size in the range of 5 to 100 bases.

[0308] Implementation Scheme 27. The kit according to Implementation Scheme 25 or 26, the kit further comprises:

[0309] (a) a first gel sequence comprising a first active group configured to be linked to a first candidate molecule, wherein the first gel sequence is complementary to the first molecule binding sequence; and

[0310] (b) A second gel sequence comprising a second active group configured to be linked to a second candidate molecule, wherein the second gel sequence is complementary to the second molecule binding sequence.

[0311] Implementation Scheme 28. The kit according to any one of Implementation Schemes 25 to 27, the kit further comprising at least one of the first candidate molecule and the second candidate molecule.

[0312] Implementation Scheme 29. The kit according to any one of Implementation Schemes 25 to 28, wherein

[0313] (a) The first candidate molecule is linked to the first gel sequence; and

[0314] (b) The second candidate molecule is linked to the second gel sequence.

[0315] Implementation Scheme 30. The kit according to any one of Implementation Schemes 25 to 29, wherein the kit further comprises a third candidate molecule.

[0316] Implementation Scheme 31. The kit according to any one of Implementation Schemes 25 to 30, wherein the first spacer polynucleotide and the second spacer polynucleotide are linked to each other by a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop.

[0317] This invention provides a support, which includes:

[0318] (a) Contains the first end of the first molecule binding sequence.

[0319] (b) An intermediate nucleic acid sequence, said intermediate nucleic acid sequence comprising:

[0320] (i) A folded sequence comprising at least one barcode, a sequence A at the 5' end

[112] and a sequence B at the 3' end

[113] , wherein sequences A and B are distinct and non-complementary.

[0321] (ii) A double-stranded nucleic acid sequence comprising a sequence complementary to the folded sequence, the sequence being juxtaposed at one end with a sequence A'

[501] complementary to sequence A and a sequence B'

[502] complementary to sequence B, and

[0322] (c) The second end contains the second molecule binding sequence.

[0323] In a preferred embodiment, the double-stranded nucleic acid sequence comprising a sequence complementary to the folded sequence contains a loop.

[0324] This invention includes a support.

[0325] - A folded sequence containing at least one barcode binds only to a double-stranded nucleic acid sequence containing a sequence complementary to the sequence folded into a hairpin structure (holiday structure) via A-to-A' and B-to-B' binding.

[0326] - A form in which a sequence folded into a hairpin structure containing at least one barcode is hybridized (invasive, final structure) with a double-stranded nucleotide containing a sequence complementary to the sequence folded into a hairpin structure.

[0327] The increase in scaffold length (and therefore the increase in the amplitude of Brownian noise) allows for the measurement of, for example, subtle interactions between two proteins.

[0328] Another primary objective provided herein is to provide a first candidate molecule linked to a first gel sequence, wherein the first gel sequence hybridizes with a first molecule-binding sequence; and a second candidate molecule linked to a second gel sequence, wherein the second gel sequence hybridizes with a second molecule-binding sequence, either alone or in combination with a scaffold according to the invention.

[0329] The first and second candidate molecules, respectively linked to the first and second gel sequences, can be any candidate molecules, preferably candidate molecules that bind to each other and / or bind to another molecule. Therefore, the scaffold of the present invention can be used to identify binding molecules for quantitative binding, etc.

[0330] In some embodiments, the scaffold of the present invention is provided, wherein the double-stranded nucleic acid complementary to the folded sequence has 10 to 3000 bases, advantageously 100 bases.

[0331] The scaffold of the present invention provides a molecular combination that specifically binds to barcodes, which is advantageously an antibody specific to modified nucleotides, particularly an antibody specific to epigenetically modified nucleotides.

[0332] In a particular embodiment, the scaffold of the invention is provided, wherein the barcode comprises at least one modified base, advantageously methylated cytosine, advantageously at least one SEQ ID NO: 3, wherein the second cytosine is epigenetically modified in Escherichia coli, or wherein the second cytosine is methylated in Escherichia coli.

[0333] Alternatively, base modifications can be obtained in vitro, and the modified bases can be inserted into the hair clip in vitro.

[0334] First, the nucleotide sequence of the scaffold of the present invention is assembled such that A hybridizes with A' and B hybridizes with B'; then, the strands of the double-stranded nucleic acid sequence can hybridize with the folded sequence when the folded sequence unfolds (opens). This requires pulling the folded structure with a force in the range of 1 pN to 25 pN.

[0335] This invention provides another primary objective:

[0336] Device or platform, comprising:

[0337] According to the invention, the first end is attached to a bead, advantageously a magnetic bead, and the second end is attached to the bottom surface of the device in a conductive solution.

[0338] - The force-applying mechanism, advantageously, is a magnet.

[0339] - A detection system for real-time detection of bead positions.

[0340] This invention provides:

[0341] Device or platform, including

[0342] - A chamber with a bottom surface,

[0343] - The force-applying mechanism, advantageously, is a magnet.

[0344] - A detection system for real-time detection of bead positions.

[0345] In addition, kits containing beads (advantageously magnetic beads), scaffolds of the present invention containing barcodes consisting of the number and position of modified bases, antibodies specific to the modified bases, conductive solutions, and a set of gel sequences that can be glued to one or more candidate molecules of interest.

[0346] Therefore, the present invention provides a method for preparing the scaffold of the present invention by first detecting the number and position of modified bases (barcodes) in a folded sequence attached to the bottom surface of beads and the device. This is achieved by measuring the bead position in real time while simultaneously performing the following:

[0347] 1) Apply force to move the beads to unfold the fold sequence.

[0348] 2) Stop the force and refold the fold sequence.

[0349] 3) Contact the refolded or unfolded folded sequence with an antibody specific for the modified bases.

[0350] 4) Apply force to move the beads.

[0351] Decode the number and position of modified bases from the position of the beads.

[0352] Therefore, the present invention provides a method for preparing the stent of the present invention, the method further comprising:

[0353] - The step of contacting a folded sequence with a decoded barcode having a double-stranded nucleic acid sequence, the folded sequence comprising sequence A

[112] at the 5' end and sequence B

[113] at the 3' end, sequences A and B being distinct and non-complementary, the double-stranded nucleic acid sequence comprising a sequence complementary to the folded sequence, the folded sequence being juxtaposed at one end with sequence A'

[501] complementary to sequence A and sequence B'

[502] complementary to sequence B.

[0354] - This leads to A combining with A', and B combining with B.

[0355] - Applying a force of 5pN to 25pN to open the folded sequence containing the decoded barcode triggers the hybridization of the double-stranded nucleotide strands with the folded sequence.

[0356] Hybridization (strand invasion) of double-stranded nucleic acid strands with folded sequences is measured by detecting bead position jumps caused by increased scaffold length.

[0357] As another objective, the present invention provides a kit comprising any one of the scaffolds of the present invention, the scaffold containing a barcode, and the barcode containing at least one modified base, an antibody specific to the modified base of the barcode in the scaffold, and a conductive solution.

[0358] The kit may also include double-stranded nucleotides containing sequences complementary to the folded sequence or groups of different sizes thereof.

[0359] The kit includes up to 20, 50, 100, 200, 500, 1000, 2000, 5000, 10000, 20000, 50000, 100000, 200000, 50000, 100000, 200000, 500000, 1000000 scaffolds of the present invention, advantageously 10000 scaffolds.

[0360] As another objective, the present invention provides a kit comprising beads, any one of the scaffolds of the present invention, the scaffold comprising a barcode and the barcode comprising at least one modified base, an antibody specific to the modified base of the barcode in the scaffold, and a conductive solution.

[0361] As another objective, the present invention provides a kit comprising beads, any one of the scaffolds of the present invention, the scaffold comprising a barcode and the barcode comprising at least one modified base, an antibody specific to the modified base of the barcode in the scaffold, a double-stranded nucleotide sequence comprising a sequence complementary to the folded sequence, and a conductive solution.

[0362] The device, scaffold, and reagent kit of the present invention are used for binding assays.

[0363] The device, scaffold, or kit of the present invention is used for binding assays, and is advantageously used for real-time binding assays.

[0364] Example

[0365] To better understand this disclosure and its many advantages, the following embodiments are given in an illustrative manner, without limiting the scope of this disclosure.

[0366] Example 1. Identification of hairpin nucleic acids

[0367] A first hairpin nucleic acid precursor sequence containing multiple distinct CCwGG (SEQ ID NO: 3) sequences spaced within a fragment or randomly generated sequence was synthesized and cloned into a plasmid for propagation in bacteria (Escherichia coli). The CCwGG (SEQ ID NO: 3) sequence was methylated in vivo by E. coli DCM methyltransferase. The plasmid was isolated from the bacteria using a commercial plasmid extraction kit, and 5 μg of plasmid was digested with BsaI. The digested fragment containing approximately 300 base pairs of spaced methylated cytosine was recovered by agarose gel electrophoresis. The hairpin nucleic acid was then ligated to the fragment with a first terminal and a first linker sequence (5' to 3'), a first Y-forming polynucleotide sequence (SEQ ID NO: 11) containing biotin at the 5' end, a second linker sequence encoding the hairpin nucleic acid, a second molecular binding sequence, a second Y-forming polynucleotide sequence (SEQ ID NO: 8) containing a second adaptor 107, and a loop (SEQ ID NO: 7). The final hairpin nucleic acid was purified by gel electrophoresis. The hairpin nucleic acid was then attached to MyOne beads (1 μm paramagnetic beads from Dynabead) using biotin present at the first end to prepare the anchored hairpin nucleic acid. The anchored hairpin nucleic acid was then immobilized to a feature portion on the bottom surface of the flow cell, wherein the feature portion contained an oligonucleotide sequence (SEQ ID NO:9) complementary to the second adaptor.

[0368] To demonstrate the ability to simultaneously detect multiple interactions, seven plasmids were mixed, each containing a different barcode (different positions of methylated cytosine, see [link]). Figure 4 Examples of possible barcodes (SEQ ID NO: 25-32). Plasmids were mixed at equimolar concentrations and treated together in the same test tube to prepare hairpin nucleic acids as described above. The hairpin nucleic acids were then positioned between the bead and the characteristic portion of the flow cell. An antibody fragment (Fab) targeting 5-mC (the Fab fragment is derived from a clone ICC / IF from Diagenode) was then injected into the flow cell. When force was applied, the clogging pattern of each bead was observed based on the binding of the antibody to the methylated cytosine of the hairpin nucleic acid. At least 100 test cycles were performed to detect the position of the methylated cytosine of all hairpin nucleic acids. Each clogging pattern is unique for a specific barcode. Figure 9 An exemplary trace showing a blockage pattern of one of the hairpin nucleic acids is illustrated, and Figure 10 The seven sequences are shown classified according to their barcodes. The method described herein provides that hairpin nucleic acids can be correctly assigned to one of the sequences, and they can be demultiplexed by the method. Therefore, this method can be used to decode the barcodes of hairpins required to simultaneously detect multiple interactions.

[0369] Example 2. Preparation of nucleic acid scaffolds

[0370] To demonstrate the ability to generate a scaffold with a spacer polynucleotide sequence (SEQ ID NO: 33), a hairpin nucleic acid corresponding to SEQ ID NO: 32 was prepared as described in Example 1 and immobilized between a bead and a feature portion on the bottom surface of a flow cell. In parallel, a spacer polynucleotide was prepared by linking a first hairpin nucleic acid precursor digested with BsaI to two specific Y-shaped forming polynucleotide sequences at one end and a spacer loop at the other end, wherein the two polynucleotide sequences comprise: (a) a first Y-shaped forming polynucleotide sequence (5' to 3') encoding a first linker sequence, and (b) a second Y-shaped forming polynucleotide sequence (5' to 3') encoding a second linker sequence. Once purified on an agarose gel, the specific spacer polynucleotide was injected into the flow cell to form a 4-way link based on the hairpin nucleic acid polynucleotide sequence, which was identified by decoding a barcode using the method provided in Example 1.

[0371] This allows anchoring spacer polynucleotides to form a 4-way link (also known as a holiday link) with the hairpin nucleic acid. A 5 pN force is then applied to the bead to dislodge the holiday link and induce strand invasion. The bead's position is recorded during injection. After dislodgement of the holiday link, the bead's Z position will shift according to the size of the hairpin following successful invasion. In this example, successful invasion corresponds to a bead position shift of approximately 200 nm, which corresponds to the length of the dsDNA adapter (600 bp / bp = 180 nm at 0.3 nm). Figure 12 An exemplary record shows the location of the beads before and after chain intrusion.

[0372] Example 3. Used to determine the binding dynamics between two substrates forming a non-homologous end-linked (NHEJ) complex. Mechanical system .

[0373] The systems described in Examples 1 and 2 were used to determine the binding kinetics between two substrates and proteins that form a non-homologous end-joint (NHEJ) complex. The NHEJ complex recognizes double-stranded blunt ends and repairs these damages in cells. Therefore, two substrates were prepared, each containing a single-stranded carrier polynucleotide and a candidate molecule containing double-stranded blunt ends, wherein the single-stranded carrier polynucleotide is complementary to the molecular binding sequence of the nucleic acid scaffold. Double-stranded blunt ends were prepared by annealing two oligonucleotides (SEQ ID NO: 14 and 15, and SEQ ID NO: 17 and 18, each 10 μM in PBS) together twice.

[0374] Because each proposed sequence contains gel sequences of both a first molecular binding sequence and a second molecular binding sequence, the substrate can be directed to specific sites on the nucleic acid scaffold. Therefore, the two substrates of the NHEJ complex are anchored to the two molecular binding sequences of the nucleic acid scaffold to form a screening nucleic acid scaffold. Excess substrate is washed away. A reference elongation length of the screening nucleic acid scaffold is determined by applying a 2pN force in the absence of a substrate.

[0375] Next, the KU70 / 80 complex and APFL protein, two candidate proteins, were injected together into PBS 1X and then into a flow cell. Flow was stopped and force cycling experiments were initiated. Blockage traces were recorded at 0.1 pN and 2 pN. At 0.1 pN, the screening scaffold was unstructured, allowing the two blunt ends to approach closely. If no protein was present in the flow cell to hold the two blunt ends together, increasing the force to 2 pN resulted in complete stretching of the screening scaffold. Similarly, if KU70 / 80 or APFL protein was loaded independently, the scaffold would stretch completely at 2 pN, indicating that they alone could not promote binding interactions between the two blunt ends. However, when both proteins were injected together, one or more transient blockages of the screening scaffold were observed at 2 pN until the interactions were disrupted and the screening scaffold was fully stretched. Figure 14 The presence of one or more transient blockages indicates that KU70 / 80 and APFL together form a complex on two substrates with double strands. Because this method is a non-destructive process, force cycling was repeated multiple times to further probe the interactions.

[0376] Experiments show that this system can be used to determine K by testing protein concentrations at various levels. on (The number of cycles of interaction observed), and K under a specific force. off (How long does it take to break the complex?) K on and K off It can be used to calculate Kd.

[0377] Example 4. Determining the binding phase between two substrates forming a non-homologous end-linked (NHEJ) complex. Interacting systems .

[0378] The systems described in Examples 1 and 2 were used to determine the binding interaction between blunt-ended substrates located on the scaffold and proteins forming non-homologous end join (NHEJ) complexes. This experiment was performed largely similarly to the method described in Example 3, except that a constant force was applied to probe the binding interaction between the two blunt ends instead of force cycling. In short, a reference amplitude of the Brownian motion displacement on the Z-axis of the nucleic acid scaffold was determined by applying a constant force. This roughly corresponds to the size of the DNA scaffold (approximately 180 nm for a 600 bp scaffold in this example). Next, a combination of KU70 / 80 and APFL was injected into a flow cell containing the nucleic acid scaffold. The interaction between the blunt-ended DNA substrate and the protein complex produced a screening nucleic acid scaffold ( Figure 15B Throughout the experiment, the amplitude of the Brownian motion of the nucleic acid screening scaffold was determined under constant force. Over time and as the NHEJ complex formed on the blunt-ended substrate, the amplitude of the Brownian motion decreased sharply to only a few nanometers due to the shortening of the scaffold. The amplitude of the Brownian motion remained low until the interaction was disrupted. The experiment demonstrates that the system can detect interactions even under low forces (<5 pN) while maintaining a constant force.

[0379] Next, K was determined by counting the number of observed events relative to the concentration of the NHEJ complex. on Furthermore, the K value of this interaction can be measured by considering the average time of the interaction between the NHEJ complex and all detected events. off .

[0380] Example 5. A system for determining the binding interaction between restriction enzymes and T4 DNA ligase.

[0381] The systems described in Examples 1 and 2 were used to determine the activities of restriction enzymes and T4 DNA ligase. Two candidate molecules were used, comprising: (a) a first candidate molecule containing a single-stranded polynucleotide complementary to the binding sequence of the first molecule and a double-stranded portion containing restriction site recognition sequences (BsaI, GGTCTC (SEQ ID NO: 19 and 20)) and a 5' overhang TATC; and (b) a second candidate molecule containing a single-stranded polynucleotide complementary to the binding sequence of the second molecule and a double-stranded portion terminated at 5' by a tetrabase overhang sequence (GAEA (SEQ ID NO: 21 and 22)). A reference elongation length of the nucleic acid scaffold was determined by applying a constant force of 0.1 pN in the absence of the candidate molecules. Next, a screening nucleic acid scaffold was generated by anchoring the two candidate molecules to the nucleic acid scaffold. Then, T4 DNA ligase was injected into a flow cell containing the screening nucleic acid scaffold to generate a screening nucleic acid scaffold. The elongation length of the screening nucleic acid scaffold was determined by applying a constant force of 0.1 pN, and the amplitude of Brownian motion was determined. No change in Brownian motion amplitude indicates that no connection has occurred between the two candidate molecules. In contrast, when a connection occurs, the amplitude of the Brownian motion will decrease due to the shortening of the scaffold, indicating a successful connection between the two candidate molecules. One indication of a successful connection is that the scaffold cannot even be stretched to the reference elongation length with more than 5 pN.

[0382] This experiment also provides a roadmap for determining reaction kinetics by varying the concentration of T4 DNA ligase and determining the time taken to ligate two substrate molecules.

[0383] This experiment also provides a roadmap for determining the kinetics of restriction enzymes (BsaI). For example, the restriction enzyme can be injected into a flow cell containing a linked candidate molecule. Digestion of the linked substrate will cause a sudden jump of approximately 200 nm in the Z-position of the bead, and the elongation length will return to the reference elongation length.

[0384] Example 6. A system for determining binding interactions between two proteins.

[0385] The systems described in Examples 1 and 2 were used to determine the binding interaction between two candidate molecules (proteins). In short, a reference elongation length of the nucleic acid scaffold present in the flow cell was determined under constant force. Next, a single-stranded polynucleotide (SEQ ID NO: 23) containing a complementary sequence of the first molecule binding sequence and an amine group at the 5' end was covalently conjugated (via EDC esterification) to the candidate protein (RBD protein from the SARV-CoV2 virus). In parallel, a second single-stranded polynucleotide (SEQ ID NO: 34) containing a complementary sequence of the second molecule binding sequence and an amine group at the 3' end was covalently conjugated (via EDC esterification) to the candidate protein (Ace2 receptor). The first candidate molecule conjugated with the first polynucleotide and the second candidate molecule covalently conjugated with the second polynucleotide were sequentially injected into the flow cell to anchor them to the nucleic acid scaffold and form a screening nucleic acid scaffold. A force of 2 pN was then applied to determine the maximum elongation length. The force was then reduced to 0.1 pN or 0.01 pN to allow the two proteins to interact. If there is a binding interaction between the two candidate molecules, the observed elongation will be less than the full elongation of the screening nucleic acid scaffold when the force is increased back to 2pN. If there is no binding interaction between the two candidate molecules or their binding interaction is weaker than the applied force, the nucleic acid scaffold will be fully stretched. This experiment provides the following roadmap: (a) conducting experiments under different forces; (b) conducting experiments under different buffer conditions; and (c) determining the strength of the interaction between the two proteins.

[0386] Example 7. A system for determining binding interactions between proteins and aptamers.

[0387] The systems described in Examples 1 and 2 were used to determine the binding interaction between two candidate molecules (protein and aptamer). In short, a reference elongation length of the nucleic acid scaffold present in a flow-through cell was determined under constant force. Two candidate molecules were engineered, comprising: (a) a single-stranded polynucleotide (SEQ ID NO: 23) containing a complementary sequence to the first molecule binding sequence and an amine group at the 5' end, coupled to a candidate protein (RBD protein from the SARV-CoV2 virus) via covalent conjugation (via EDC esterification); and (b) a second candidate molecule (aptamer) containing a sequence complementary to the second molecule binding sequence and a polynucleotide sequence encoding the aptamer targeting the RBD protein (SEQ ID NO: 10). The first and second candidate molecules were then sequentially injected into the flow-through cell to anchor them to the nucleic acid scaffold and form a screening nucleic acid scaffold. A constant force of 2 pN was applied to determine the maximum elongation length of the screening nucleic acid scaffold. The force was then reduced to allow the two candidate molecules to interact. If there is a binding interaction between the two candidate molecules, the displacement of the bead will be less than the maximum length when the force is increased to 2 pN. If there is no binding interaction between the two candidate molecules or their binding interaction is weaker than the applied constant force, a maximum extension is observed. The force cycle can be repeated to determine the binding kinetics between the two candidates.

[0388] Example 8. A system for determining the binding interaction between a bispecific antibody and a candidate antigen.

[0389] The systems described in Examples 1 and 2 were used to determine the binding interactions between the bispecific antibody and two antigens. Briefly, a reference trace of the nucleic acid scaffold was recorded by applying a constant force of 0.1 pN for two minutes to determine the amplitude of Brownian motion. Next, two candidate molecules were sequentially injected into a flow-through cell containing the nucleic acid scaffold to anchor them to the scaffold and form a screening scaffold. The two candidate molecules included: (a) a first candidate molecule (polynucleotide) containing a sequence complementary to the binding sequence of the first molecule (first gel sequence) and a single-chain region having a methylated cytosine (5-methylcytosine) (SEQ.ID NO: 19); and (b) a second candidate molecule (polynucleotide) containing a sequence complementary to the binding sequence of the second molecule (second gel sequence) and a single-chain region having a methylated cytosine (5-methylcytosine) (SEQ.ID NO: 21). Then, a bispecific antibody against 5-mC modification (from the ICC clone of Diagenode, catalog number C15200003) was injected into the flow-through cell. Binding events were determined by recording the traces of the screening nucleic acid scaffold under the same constant force of 0.1 pN for 30 minutes. A decrease in noise at the appearance of the beads indicated a binding event.

[0390] Example 9. A system for determining the structural features of RNA

[0391] The systems described in Examples 1 and 2 are used to determine the binding interactions between two candidate molecules. In short, candidate RNA molecules with secondary structures (e.g., preQ1 structures) are engineered to include a sequence complementary to the first molecule-binding sequence at the 5' end and a sequence complementary to the second molecule-binding sequence at the 3' end. The candidate molecule is then injected into a flow-through cell to anchor it to a nucleic acid scaffold, thereby forming a bridge between the first and second molecule-binding sequences. When a force is applied to the system, it causes the RNA molecule to unfold. Next, a third candidate molecule capable of binding the RNA secondary structure (e.g., a PreQ1 ligand) is injected into the flow-through cell at different concentrations. Next, ramp cycling, force cycling, or applying a constant step force are performed to determine the binding kinetics of the third candidate molecule to the RNA structure and to determine whether the binding of the third candidate molecule is stable or unstable to the structure. This experiment provides a roadmap for determining multiple different RNA secondary structures within the same flow-through cell without altering the cassette.

[0392] The experiment provides a roadmap for determining the effects of one or more candidate molecules on the secondary structure of RNA, for example, by injecting one or more candidate molecules into a flow cell and repeating ramp cycles, force cycles, or applying step constant forces as described above.

[0393] Example 10. Identification of hairpin nucleic acids

[0394] The sequence of the first hairpin nucleic acid precursor, containing multiple CCwGG (SEQ ID NO: 3) sequences spaced within the fragment, was synthesized and cloned into a plasmid for propagation in bacteria (Escherichia coli). The CCwGG (SEQ ID NO: 3) sequence was methylated in vivo by E. coli DCM methyltransferase. The plasmid was isolated from the bacteria using a commercial plasmid extraction kit, and 5 μg of plasmid was digested with BsaI. The digested fragment containing approximately 300 base pairs of spaced methylated cytosine was recovered by agarose gel electrophoresis. Then, a first Y-forming polynucleotide sequence (SEQ ID NO: 11) encoding the hairpin nucleic acid, a second linker sequence, a second molecular binding sequence, a second Y-forming polynucleotide sequence (SEQ ID NO: 8) encoding the hairpin nucleic acid, and a loop (SEQ ID NO: 7) were ligated onto the fragment to form the hairpin nucleic acid. The final hairpin nucleic acid was purified by gel electrophoresis. The hairpin nucleic acid was then attached to MyOne beads (1 μm paramagnetic beads from Dynabead) using biotin present at the first end to prepare the anchored hairpin nucleic acid. The anchored hairpin nucleic acid was then immobilized to a feature portion on the bottom surface of the flow cell, wherein the feature portion contained an oligonucleotide sequence (SEQ ID NO: 9) complementary to the second adaptor.

[0395] To demonstrate the ability to simultaneously detect multiple interactions, seven plasmids were mixed, each containing a different barcode (different positions of methylated cytosine, see [link]). Figure 4 (Examples of possible barcodes). Plasmids were mixed at equimolar concentrations and treated together in the same test tube to prepare hairpin nucleic acids as described above. The hairpin nucleic acids were then positioned between the bead and the characteristic portion of the flow cell. An antibody fragment (Fab) targeting 5-mC (derived from a cloned ICC / IF from Diagenode) was then injected into the flow cell. When force was applied, the clogging pattern of each bead was observed based on the binding of the antibody to the methylated cytosine of the hairpin nucleic acid. At least 100 test cycles were performed to detect the position of the methylated cytosine of all hairpin nucleic acids. Each clogging pattern is unique for a specific barcode. Figure 13 A and Figure 13 B illustrates an exemplary trace showing a clogging pattern of one of the hairpin nucleic acids. The methods described herein provide that hairpin nucleic acids can be correctly assigned to one of the sequences, and that they can be demultiplexed by the methods described. Therefore, the method can be used to decode barcodes of hairpins required to simultaneously detect multiple interactions.

[0396] Example 11. Preparation of nucleic acid scaffolds

[0397] Six hairpin nucleic acids attached to the beads and the characteristic portion of the flow cell bottom surface were prepared as described in Example 1. In parallel, a spacer polynucleotide was prepared by linking a first hairpin nucleic acid precursor digested with BsaI to two specific Y-shaped forming polynucleotide sequences at one end and a spacer loop at the other end, wherein the two polynucleotide sequences comprise: (a) a first Y-shaped forming polynucleotide sequence (5' to 3') encoding a first linker sequence, and (b) a second Y-shaped forming polynucleotide sequence (5' to 3') encoding a second linker sequence. Once purified on an agarose gel, the specific spacer polynucleotide was injected into the flow cell to form a 4-way link based on the hairpin nucleic acid polynucleotide sequence, which was identified by decoding the barcode using the method provided in Example 1.

[0398] This allows anchoring spacer polynucleotides to form a 4-way link (also known as a holiday link) with the hairpin nucleic acid. A 5 pN force is then applied to the bead to dislodge the holiday link and induce strand invasion. The bead's position is recorded during injection. After dislodgement of the holiday link, the bead's Z position will shift according to the size of the hairpin following successful invasion. In this example, successful invasion corresponds to a bead position shift of approximately 200 nm, which corresponds to the length of the dsDNA adapter (600 bp / bp = 180 nm at 0.3 nm). Figure 23 An exemplary record shows the location of the beads before and after chain intrusion. Figure 24 The chain intrusion efficiency in six hairpin nucleic acids containing universal barcode sequences is shown. Figure 24 Analysis showed that the single nucleotide polymorphism difference between the universal chain intrusion and the barcode sequence (SEQ ID NO: 38) (due to the sequence CawGG (SEQ ID NO: 54) changing to CCwGG (SEQ ID NO: 3)) does not affect the effectiveness of the chain intrusion. This allows us to use single-chain intrusion hairpins for all barcodes.

[0399] Example 12. Used to determine the binding dynamics between two substrates forming a non-homologous end-linked (NHEJ) complex. Mechanical system .

[0400] The systems described in Examples 1 and 2 were used to determine the binding kinetics between two substrates and proteins that form a non-homologous end joiner (NHEJ) complex. The NHEJ complex recognizes double-stranded blunt ends and repairs these damages in cells. Therefore, two substrates were prepared, each containing a single-stranded carrier polynucleotide and a candidate molecule containing double-stranded blunt ends, wherein the single-stranded carrier polynucleotide is complementary to the molecular binding sequence of the nucleic acid scaffold. Two double-stranded blunt-end adaptors (SEQ ID NO: 14 and 15, and SEQ ID NO: 17 and 18, each in a 10 µM buffer containing 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM DTT at pH 7.9, 25 °C) were prepared by annealing the two oligonucleotides together in two separate tubes.

[0401] Because the proposed system is based on a protein complex that recognizes blunt-ended DNA, the substrate can be directed to a specific location on the nucleic acid scaffold. Therefore, two substrates from the NHEJ complex are anchored to two molecularly binding sequences on the nucleic acid scaffold to form a screening scaffold. Excess substrate is washed away. A reference elongation length for the screening scaffold is determined by applying force cycles at three steps: 0.01 N, 2 N, and 20 pN forces in the absence of substrate.

[0402] Next, the two candidate proteins, the KU70 / 80 complex and the APFL protein, were added to a buffer solution containing 20 mM Hepes-KOH (pH 7.8), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 0.5 mg / mL BSA, and injected into a flow cell. Flow was stopped and a force cycling experiment was initiated. The force cycling was recorded, showing the force variations between three plateaus: 0.01 pN, 2 pN, and 20 pN. Figure 25A At a force of 0.01 pN, the screening nucleic acid scaffold is unstructured, allowing the two blunt ends to approach closely. Increasing the force to 2 pN will result in complete stretching of the screening nucleic acid scaffold if no protein is present in the flow cell to hold the two blunt ends together. Similarly, if only KU70 / 80 or APFL proteins are loaded independently, the scaffold will stretch completely at 2 pN, indicating that they alone cannot promote binding interactions between the two blunt ends. However, when both proteins are injected together, one or more transient blockages of the screening nucleic acid scaffold are observed at 2 pN until the interaction is disrupted and the screening nucleic acid scaffold is fully stretched. The presence of one or more transient blockages indicates that KU70 / 80 and APFL together form a complex on both substrates of the double strand (…). Figure 25B Because this method is a non-destructive process, the force cycle is repeated multiple times to further explore the interactions.

[0403] Experiments show that this system can be used to determine K by testing protein concentrations at various levels. on (The number of cycles of interaction observed), and K under a specific force. off (How long does it take to break the complex?) K on and K off It can be used to calculate Kd.

[0404] Example 13. System for determining the binding interaction between two substrates ACE2 and SARS-CoV2 RBD .

[0405] The systems described in Examples 1 and 2 are used to determine the binding interactions between blunt-ended substrates located on a scaffold and proteins that form non-homologous end junction (NHEJ) complexes. Figure 26A A graphical representation of how the binding dynamics between two candidate molecules can be determined under constant low force is shown. The experiment is largely similar to the method described in Example 3, except that a constant force is applied to probe the binding interaction between the two proteins instead of force cycling. In short, a reference amplitude of the Brownian motion displacement on the Z-axis of the nucleic acid scaffold is determined by applying a constant force. This roughly corresponds to the size of the DNA scaffold (approximately 180 nm for a 600 bp scaffold in this example, or approximately 270 nm for a 900 bp scaffold). Next, ACE2 labeled with gel nucleotides and RBD with a second gel oligonucleotide are injected into a flow cell containing the nucleic acid scaffold. The interaction between the two proteins produces a screening nucleic acid scaffold. Throughout the experiment, the amplitude of the Brownian motion of the screening nucleic acid scaffold is determined under low constant force. Over time and with the formation of the ACE2-RBD complex, the amplitude of the Brownian motion decreases sharply to only a few nanometers due to the shortening of the scaffold (from 900 bp to 300 bp). The amplitude of the Brownian motion remains low until the interaction is disrupted. Experiments show that the system can detect interactions even under low forces (<5pN) while maintaining a constant force.

[0406] Next, K was determined by counting the number of observed events relative to the concentration of the NHEJ complex. on Furthermore, the K value of this interaction can be measured by considering the average time of the interaction between the NHEJ complex and all detected events. off .

[0407] Example 14. A system for determining the binding interaction between a bispecific antibody and a candidate antigen.

[0408] The systems described in Examples 1 and 2 were used to determine the binding interactions between the bispecific antibody and two antigens. Briefly, a reference trace of the nucleic acid scaffold was recorded by applying a constant force of 0.01 pN for two minutes to determine the amplitude of Brownian motion. Figure 26BNext, two candidate molecules were sequentially injected into a flow cell containing a nucleic acid scaffold to anchor them to the scaffold and form a screening nucleic acid scaffold. The two candidate molecules included: (a) a first candidate molecule (antigen) covalently attached to an oligonucleotide containing a sequence complementary to the binding sequence of the first molecule (SEQ.ID NO: 19); and (b) a second candidate molecule (antigen) covalently attached to an oligonucleotide containing a sequence complementary to the binding sequence of the second molecule (SEQ.ID NO: 21). Then, a bispecific antibody was injected into the flow cell. Binding events were determined by recording the traces of the screening nucleic acid scaffold at the same constant force of 0.01 pN for 5 minutes. The force was then increased to 20 pN to remove all interactions, and the force was reduced back to 0.01 pN. An exemplary trace is shown in... Figure 26C As shown in the diagram. Because this is a non-destructive measurement, the cycle can be repeated as many times as needed to obtain high-quality data. The reduction in noise from the appearance of the beads indicates a binding event.

[0409] Example 15. Determining the binding interaction system between two proteins via the presence of PROTAC or molecular glue Unification

[0410] The systems described in Examples 1 and 2 were used to determine the binding interaction between two candidate molecules (proteins) in the presence of a molecular gel. In short, a reference elongation length of the nucleic acid scaffold present in the flow cell was determined under constant force. Next, a single-stranded polynucleotide (SEQ ID NO: 23) containing a complementary sequence of the first molecule binding sequence and an amino group at the 5' end was covalently conjugated (via EDC esterification) to a candidate protein (CRBN forming an E3 ligase complex and its binding partner DDB1). In parallel, a second single-stranded polynucleotide (SEQ ID NO: 34) containing a complementary sequence of the second molecule binding sequence and an amino group at the 3' end was covalently conjugated (via EDC esterification) to a candidate protein (a novel GSTP1 substrate of CRBN). The first candidate molecule conjugated with the first polynucleotide and the second candidate molecule covalently conjugated with the second polynucleotide were sequentially injected into the flow cell to anchor them to the nucleic acid scaffold and form a screening nucleic acid scaffold. Figure 27 and Figure 28A force of 2 pN was then applied to determine the maximum elongation length. The force was then reduced to 0.01 pN to allow the two proteins to interact. If there was a binding interaction between the two candidate molecules, the observed elongation was less than the full extension of the screening nucleic acid scaffold when the force was increased back to 2 pN. If there was no binding interaction between the two candidate molecules or their binding interaction was weaker than the applied force, the nucleic acid scaffold would be fully stretched. The two proteins were not considered to have a native interaction. However, interactions were observed when a compound called a proteolytically targeted chimera (PROTAC) or a molecular gel was added to the flow cell. Similar results were also observed with 10 nM of the compound CC885 or 1 nM of thalidomide. Various concentrations of the compound can be used to measure the Kon and Koff interactions in the presence of the compound.

[0411] Similar experiments were also conducted on another novel substrate of CRBN E3 ligase, IKZF1 (IKAROS transcription factor), in the presence of 1 nM pomalidomide, where interactions were observed. Figure 29 ).

[0412] Example 16. Multiple interactions between a protein and two other proteins

[0413] Multiple interactions between ACE2 and RBD proteins (wild-type RBD and RBDΔ mutant) were determined. In short, two hairpin nucleic acids were immobilized between the bead and the feature region to determine multiple interactions: (1) the first hairpin nucleic acid of SEQ ID NO: 48 was used to determine the interaction between ACE2 and wild-type RBD; and (2) the second hairpin nucleic acid of SEQ ID NO: 49 was used to determine the interaction between ACE2 and the RBDΔ mutant. Next, three gel sequences were engineered: (1) the first gel sequence of SEQ ID NO: 45 was conjugated to the ACE2 protein, which anchored the ACE2 protein to the two hairpin nucleic acids; (2) the second gel sequence of SEQ ID NO: 46 was conjugated to the wild-type RBD protein, which anchored the wild-type RBD protein to the first hairpin nucleic acid; and (3) the third gel sequence of SEQ ID NO: 47 was conjugated to the RBDΔ mutant protein, which anchored the RBDΔ mutant protein to the second hairpin nucleic acid. The identification of the barcoded molecules was performed according to Example 10, and the preparation of the nucleic acid scaffold was performed according to Example 11. RBD proteins (wild-type RBD protein and RBTΔ mutant protein) were then injected separately, and binding interactions and binding kinetics were measured to assess the specificity of anchoring the protein to the specific barcoded molecule. The results of the binding interaction between ACE2 and the RBD protein are provided in... Figure 30 middle.

[0414] While exemplary embodiments have been shown and described herein, such embodiments are provided by way of example only. Many variations, modifications, and substitutions are within the scope of this disclosure. 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 this disclosure and thereby cover the methods and structures within the scope of these claims, as well as their equivalents.

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Claims

1. A nucleic acid scaffold for determining binding interactions between a first candidate molecule and a second candidate molecule, wherein the nucleic acid scaffold comprises: (a) A continuous polynucleotide sequence, said continuous polynucleotide sequence comprising: (i) Attached to a first end of a bead, wherein the first end contains a first molecular binding sequence. (ii) A second end attached to the bottom surface of the device, wherein the second end comprises a second molecular binding sequence, and (iii) The intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises: (I) The first lead containing the barcode forms a sequence. (II) A second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence hybridizes with the second lead-forming sequence, and (III) A loop connecting the first lead-forming sequence and the second lead-forming sequence; (b) a first spacer polynucleotide, the first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and (c) A second spacer polynucleotide, wherein the second spacer polynucleotide has a polynucleotide sequence complementary to the second lead-forming sequence. The first spacer polynucleotide and the second spacer polynucleotide hybridize with the middle portion, and The nucleic acid scaffold requires a force of 0.1 pN to 10 pN to be applied to the beads along an axis perpendicular to the bottom surface of the device to stretch the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold.

2. The nucleic acid scaffold of claim 1, wherein the barcode sequence comprises one or more modified nucleotides.

3. The nucleic acid scaffold according to any one of claims 1 to 2, wherein the intermediate portion further comprises a first ligation sequence and a second ligation sequence, wherein the first ligation sequence is located between the first molecule-binding sequence and the first lead-forming sequence, and wherein the second ligation sequence is located between the second molecule-binding sequence and the second lead-forming sequence, and wherein the first ligation sequence and the second ligation sequence are not complementary to each other.

4. The nucleic acid scaffold according to any one of claims 1 to 3, wherein the first spacer polynucleotide and the second spacer polynucleotide are connected to each other by a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop.

5. The nucleic acid scaffold according to any one of claims 1 to 4, wherein the first molecular binding sequence and the second molecular binding sequence each independently comprise a hairpin nucleic acid having a size in the range of 5 to 100 bases.

6. A nucleic acid screening scaffold, the nucleic acid screening scaffold comprising: (a) A nucleic acid scaffold according to any one of claims 1 to 5; (b) ligating a first candidate molecule to a first gel sequence, wherein the first gel sequence hybridizes with a first molecule-binding sequence; and (c) A second candidate molecule is attached to a second gel sequence, wherein the second gel sequence hybridizes with the binding sequence of the second molecule.

7. An apparatus comprising: (a) A chamber disposed within the device, wherein the chamber includes a bottom surface; (b) A force application mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; as well as (c) The nucleic acid scaffold according to any one of claims 1 to 5 or the screening nucleic acid scaffold according to claim 6.

8. A method for determining the binding interaction between a first candidate molecule and a second candidate molecule, the method comprising: (a) Providing a nucleic acid scaffold according to any one of claims 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device; (b) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; (d) Determine in real time the reference elongation length of the nucleic acid scaffold in response to force in the absence of the first candidate molecule and the second candidate molecule: (i) A force of 0.1 pN to 50 pN is applied to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and (ii) The change in the position of the beads along the axis is measured via a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule; (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to each other. (f) By repeating (d), determine the elongation length of the screening nucleic acid scaffold in response to the same force used in the absence of the first and second candidate molecules; and (g) Calculate the difference, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold under the applied force and the reference elongation length. A non-zero difference indicates that the binding interaction exists between the first candidate molecule and the second molecule under the applied force, and a zero difference indicates that the binding interaction does not exist between the first candidate molecule and the second candidate molecule under the applied force.

9. The method of claim 8, further comprising determining the binding kinetics of the binding interaction between the first candidate molecule and the second candidate molecule by: (h) after (g), removing the force applied to the bead by the force application mechanism, thereby causing relaxation of the screening nucleic acid scaffold; and (i) repeating (f) to (h) and calculating the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length as a function of time.

10. The method according to claim 9, further comprising: (i) Determine the K-value of the binding between the second molecule and the first candidate molecule. on Wherein K on The calculation is based on the number of cycles (f) to (h) that result in the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length.

11. The method according to claim 9, further comprising: (h) Determine the K-value of the binding between the second molecule and the first candidate molecule. off Wherein K off The calculation is based on the length of time during which the difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length exists during each cycle of repetition (f) to (h).

12. A method for determining binding interactions among a first candidate molecule, a second candidate molecule, and a third candidate molecule, the method comprising: (a) Providing a nucleic acid scaffold according to any one of claims 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device; (b) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; (d) Determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule by the following method: (i) A force of 0.1 pN to 50 pN is applied to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and (ii) The change in the position of the beads along the axis is measured via a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule; (e) The nucleic acid scaffold is brought into contact with the first candidate molecule and the second candidate molecule, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to each other; (f) By repeating (d), determine the force required to achieve the same elongation length as the reference elongation length of the screening nucleic acid scaffold, wherein the required force is in the range of 0.1 pN to 50 pN; (g) bringing the third candidate molecule into contact with the screening nucleic acid scaffold; and (h) By repeating (d), determine the elongation length of the screening nucleic acid scaffold in the presence of the third candidate molecule and in response to the same force applied in (f); and (i) Calculate the difference, wherein the difference is the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule. A non-zero difference indicates that a binding interaction exists between the third candidate molecule and the first and second candidate molecules under the applied force, and a zero difference indicates that no such binding interaction exists between them: (A) The third candidate molecule and the first candidate molecule. (B) The third candidate molecule and the second candidate molecule, or (C) The third candidate molecule under the applied force, together with the first candidate molecule and the second candidate molecule.

13. The method of claim 12, further comprising determining the binding kinetics of the binding interactions between the first candidate molecule, the second candidate molecule, and the third candidate molecule by: (j) after (i), removing the force applied to the beads by the force application mechanism, thereby causing relaxation of the test screening nucleic acid scaffold; and (k) repeating (e) to (j) and calculating the difference in the elongation length of the screening nucleic acid scaffold as a function of time in the presence and absence of the third candidate molecule.

14. The method according to claim 12, further comprising: (j) Determine the K-value of the binding of the third candidate molecule to the first and second molecules. on Wherein K on The calculation is based on the number of repetitions (h) to (j) that result in the difference between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule.

15. The method according to claim 12, further comprising: (j) Determine the K-value of the binding of the third candidate molecule with the first candidate molecule and the second candidate molecule. off Wherein K off The calculation is based on the length of time during each cycle of repetition (h) to (j) that the difference exists between the elongation length of the screening nucleic acid scaffold in the presence and absence of the third candidate molecule.

16. A method for screening binding interactions between a first candidate molecule and a plurality of second candidate molecules, the method comprising: (a) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface. (ii) Force-applying mechanism, and (iii) A plurality of nucleic acid scaffolds positioned along an axis perpendicular to the bottom surface of the chamber of the device, wherein each of the plurality of nucleic acid scaffolds comprises a nucleic acid scaffold according to any one of claims 1 to 5, wherein each of the plurality of nucleic acid scaffolds is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the device; (b) Determine the reference elongation length of each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules by the following method: (i) A force of 0.1 pN to 50 pN is applied via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device to the beads attached to each of the plurality of nucleic acid scaffolds, wherein each of the plurality of nucleic acid scaffolds is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to each of the plurality of nucleic acid scaffolds along the axis, and (ii) The change in the position of the bead along the axis is measured via a sensor to determine a reference elongation length for each of the plurality of nucleic acid scaffolds in the absence of the first candidate molecule and the plurality of second candidate molecules; (c) Contacting the plurality of nucleic acid scaffolds with the first candidate molecule linked to the first gel sequence, thereby anchoring the first candidate molecule to each of the plurality of nucleic acid scaffolds, wherein the first gel sequence is complementary to the first molecule binding sequence; (d) Contacting the plurality of nucleic acid scaffolds with the plurality of second candidate molecules each connected to a second gel sequence, thereby anchoring one of the second candidate molecules to each of the plurality of nucleic acid scaffolds, wherein the second gel sequence is complementary to the second molecule binding sequence, thereby forming a plurality of screening nucleic acid scaffolds, wherein the first candidate molecule and the second candidate molecule of each of the plurality of screening nucleic acid scaffolds are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other; (e) By repeating (d), the elongation length of each of the plurality of screening nucleic acid scaffolds in response to a force from 0.1 pN to 50 pN is determined; and (f) Calculate the difference between the reference elongation length and the elongation length of each of the plurality of screening nucleic acid scaffolds. The difference between the elongation length of the screening nucleic acid scaffold and the reference elongation length indicates that the first candidate molecule and the second candidate molecule anchored to the screening nucleic acid have a binding interaction with each other under the applied force, and the absence of the difference indicates that there is no binding interaction between the first candidate molecule and the second candidate molecule under the applied force.

17. The method of claim 16, wherein at least two nucleic acid scaffolds in the nucleic acid scaffold comprise barcode sequences located at different positions relative to each other.

18. The method of claim 16, wherein at least two nucleic acid scaffolds in the nucleic acid scaffold contain barcode sequences that are different from each other.

19. The method of claim 17 or 18, further comprising determining the identity of the consecutive polynucleotide sequences based on the barcode prior to (b).

20. The method of claim 19, wherein the identity of the consecutive polynucleotide sequences is determined by detecting the position of one or more modified nucleotides in the barcode.

21. A method for screening binding interactions between a first candidate molecule, a second candidate molecule, and a plurality of third candidate molecules, the method comprising: (a) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface. (ii) Force-applying mechanism, and (iii) A nucleic acid scaffold, the nucleic acid scaffold being positioned along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold comprises a nucleic acid scaffold according to any one of claims 1 to 5, wherein the nucleic acid scaffold is connected at one end to a bead and at the other end to a feature portion of the bottom surface of the chamber of the device; (b) The reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the second candidate molecule is determined by the following method: (iv) A force of 0.1 pN to 50 pN is applied to the beads attached to the nucleic acid scaffold via the force application mechanism along an axis perpendicular to the bottom surface of the chamber of the device, wherein the nucleic acid scaffold is configured to deploy in response to the applied force, thereby causing a change in the position of the beads attached to the nucleic acid scaffold along the axis, and (v) The change in the position of the beads along the axis is measured via a sensor to determine the reference elongation length of the nucleic acid scaffold in the absence of the first candidate molecule and the plurality of second candidate molecules; (c) Contact the nucleic acid scaffold with the first candidate molecule linked to the first gel sequence, thereby anchoring the first candidate molecule to the nucleic acid scaffold; (d) Contact the nucleic acid scaffold with the second candidate molecule linked to the second gel sequence to anchor the second candidate molecule to the nucleic acid scaffold, thereby forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule of the screening nucleic acid scaffold are positioned such that the first candidate molecule and the second candidate molecule are adjacent to each other. (e) By repeating (d), determine the force required to achieve the same elongation length as the reference elongation length of the screening nucleic acid scaffold, wherein the force is in the range of 0.1 pN to 50 pN; (f) Contact the plurality of third candidate molecules with the screening nucleic acid scaffold; (g) By repeating (b), determine the elongation length of the screening nucleic acid scaffold in response to the same force applied in (e) in the presence of at least one of the plurality of third candidate molecules; and (h) Calculate the difference between the reference elongation length and the elongation length of the screening nucleic acid scaffold. A non-zero difference indicates that a binding interaction exists between the third candidate molecule and the first and second candidate molecules under the applied force, and a zero difference indicates that no such binding interaction exists between them: (A) The third candidate molecule and the first candidate molecule. (B) The third candidate molecule and the second candidate molecule, or (C) The third candidate molecule under the applied force, together with the first candidate molecule and the second candidate molecule.

22. A method for determining the binding interaction between a first candidate molecule and a second candidate molecule, the method comprising: (a) Providing a nucleic acid scaffold according to any one of claims 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device; (b) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; (d) Determine the reference amplitude of the nucleic acid scaffold in response to Brownian noise of a force less than 0.01 pN in the absence of the first candidate molecule and the second candidate molecule; (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other. (f) By repeating (d), determine the amplitude of the screening nucleic acid scaffold in response to the Brownian noise of the same force used in the absence of the first and second candidate molecules; and (g) Identify an interaction event between the two molecules, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the molecules.

23. A method for determining binding interactions among a first candidate molecule, a second candidate molecule, and a third candidate molecule, the method comprising: (a) Providing a nucleic acid scaffold according to any one of claims 1 to 5, wherein the nucleic acid scaffold is positioned along an axis perpendicular to the bottom surface of the chamber of the device; (b) Provides an apparatus comprising: (i) a chamber disposed within the device, wherein the chamber includes a bottom surface, and (ii) A force-applying mechanism for stretching the continuous polynucleotide sequence along the axis to form the nucleic acid scaffold; (c) Providing a first candidate molecule linked to a first gel sequence and a second candidate molecule linked to a second gel sequence, wherein the first gel sequence is complementary to the binding sequence of the first molecule, and wherein the second gel sequence is complementary to the binding sequence of the second molecule; (d) Determine the reference amplitude of the nucleic acid scaffold in response to Brownian noise of a force less than 0.01 pN in the absence of the first candidate molecule and the second candidate molecule; (e) The nucleic acid scaffold is brought into contact with the first candidate molecule linked to the first gel sequence and the second candidate molecule linked to the second gel sequence, thereby attaching the first candidate molecule and the second candidate molecule to the first molecule binding sequence and the second molecule binding sequence, respectively, and forming a screening nucleic acid scaffold, wherein the first candidate molecule and the second candidate molecule are positioned along the screening nucleic acid scaffold such that the first candidate molecule and the second candidate molecule are adjacent to or in contact with each other. (f) Contact the third candidate molecule in the solution with the screening nucleic acid scaffold; (g) Determine the amplitude of the Brownian noise of the screening nucleic acid scaffold in response to the same force applied in (d) in the presence of the third candidate molecule; and (h) Identifying an event of interaction between the first molecule, the second molecule, and the third molecule, wherein the amplitude of the Brownian noise is reduced compared to the reference amplitude under the same force in the absence of the first molecule, the second molecule, and the third molecule.

24. A kit for determining binding interactions between a first candidate molecule and a second candidate molecule, the kit comprising: (a) Nucleic acid, said nucleic acid comprising: (i) a continuous polynucleotide sequence, the continuous polynucleotide sequence comprising: (I) A first end, the first end comprising: (i) a first linker for attachment to a bead, and (ii) a first molecular binding sequence; (II) A second end, comprising: (i) a second adapter for attaching the nucleic acid to the bottom surface of the device, and (ii) a second molecule binding sequence; (III) The intermediate portion between the first molecule-binding sequence at the first end and the second molecule-binding sequence at the second end, wherein the intermediate portion comprises:

1. The first lead containing the barcode forms a sequence.

2. A second lead-forming sequence complementary to the first lead-forming sequence, wherein the first lead-forming sequence hybridizes with the second lead-forming sequence, and 3. A loop connecting the first lead-forming sequence and the second lead-forming sequence; (ii) a first spacer polynucleotide, the first spacer polynucleotide having a polynucleotide sequence complementary to the first lead-forming sequence; and (iii) A second spacer polynucleotide, wherein the second spacer polynucleotide has a polynucleotide sequence complementary to the second lead-forming sequence. When the nucleic acid is attached to the bottom surface of the device as a nucleic acid scaffold, a force of 10 pN to 30 pN is required to unfold the continuous polynucleotide sequence along an axis perpendicular to the bottom surface of the device; and (b) beads containing anchoring molecules configured to bind to the first terminator of the first end of the continuous polynucleotide sequence of the nucleic acid.

25. The kit of claim 24, wherein the first molecular binding sequence and the second molecular binding sequence each independently comprise a small hairpin nucleic acid having a size in the range of 5 to 100 bases.

26. The kit according to claim 22 or 25, further comprising: (a) a first gel sequence comprising a first active group configured to be linked to a first candidate molecule, wherein the first gel sequence is complementary to the binding sequence of the first molecule; and (b) A second gel sequence comprising a second active group configured to be linked to a second candidate molecule, wherein the second gel sequence is complementary to the second molecule binding sequence.

27. The kit according to any one of claims 22 to 26, wherein the kit further comprises at least one of the first candidate molecule and the second candidate molecule.

28. The kit according to any one of claims 22 to 27, wherein the kit further comprises a third candidate molecule.

29. The kit according to any one of claims 22 to 28, wherein the first spacer polynucleotide and the second spacer polynucleotide are linked to each other by a spacer loop sequence, wherein the spacer loop sequence hybridizes with the loop.

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