Methods and systems for processing or analyzing oligonucleotide-encoded molecules

Abstract

The present disclosure provides methods and systems for determining the target activity of at least one resolved oligonucleotide encoding molecule. In one embodiment, the method includes providing a separation medium, wherein the separation medium contains at least one target molecule; and various methods for separating a mixture of at least two oligonucleotide encoding molecules by electrophoresis based on the different target activities of the oligonucleotide encoding molecule to the target molecule. The benefits of the method disclosed herein may include, but are not limited to, collecting and calculating the qualitative and quantitative data of the target activity of the encoded portion of the oligonucleotide encoding molecule to the target molecule.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 818,645, filed on March 14, 2019, the entire contents of which are hereby incorporated by reference herein. Background Art

[0003] Oligonucleotide-encoded libraries can provide a useful method for guiding combinatorial synthesis and identifying a large number of different molecules with different properties and reactivity. In general, oligonucleotide-encoded molecules can include a coding portion connected to an encoded portion, such as an oligonucleotide. In general, the coding portion is used to record or guide the combinatorial synthesis of the encoded portion, and is used to identify the structure of the encoded portion after synthesis. By analogy, the coding portion is like a molecular barcode for a 3-D printer, which tells the printer what to produce and then remains attached to identify the product after printing.

[0004] However, once libraries of millions to trillions of different oligonucleotide-encoded molecules have been synthesized, the challenge is to determine what useful properties these encoded portions may have. The synthesis and next-generation sequencing of oligonucleotide-encoded molecules has been the subject of intense academic and industrial research, leading to increasingly efficient methods for synthesizing oligonucleotide-encoded molecules, rapidly identifying their encoded portions, and determining their useful properties. Despite these advances, methods for isolating, measuring, and / or determining the utility of the encoded portions of these molecules have not kept pace.

[0005] Still need to effectively separate oligonucleotide encoding molecules based on the binding target activity of oligonucleotide encoding molecules to target molecules. Still need to eliminate or reduce false positive and false negative results. Still need to provide qualitative and / or quantitative data about the target activity of single oligonucleotide encoding molecules to target molecules. Summary of the invention

[0006] The present disclosure provides methods and systems for collecting target activity data of at least one resolved oligonucleotide encoding molecule based at least in part on different target activities of the oligonucleotide encoding molecule to the target molecule (as determined by electrophoresis and oligonucleotide sequencing). The present disclosure also provides methods and systems for separating a mixture of at least two oligonucleotide encoding molecules by electrophoresis based at least in part on different target activities of the oligonucleotide encoding molecule to the target molecule. The benefits of the methods disclosed herein may include, for example, providing qualitative and quantitative data of the target activity of the encoded portion of the oligonucleotide encoding molecule to the target molecule.

[0007] A method for determining the target activity of at least one resolved oligonucleotide encoding molecule includes providing a separation medium, wherein the separation medium contains at least one target molecule; introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules into the separation medium, wherein the at least two different oligonucleotide encoding molecules include a coding portion operably linked to at least one encoded portion; forming at least two different resolved oligonucleotide encoding molecules by separating the at least two different oligonucleotide encoding molecules into at least two separate locations in the separation medium; harvesting at least one resolved oligonucleotide encoding molecule from the at least two different resolved oligonucleotide encoding molecules by separating at least one of the at least two separate locations from the separation medium to form at least one resolved fragment; treating at least one resolved oligonucleotide encoding molecule to allow for polymerase chain reaction (PCR); amplifying at least one encoded portion of the at least one resolved oligonucleotide encoding molecule by PCR on the coding portion of the at least one resolved oligonucleotide encoding molecule; and determining the target activity of the at least one resolved oligonucleotide encoding molecule by treating at least one position and identity of at least one encoded portion of the at least one resolved oligonucleotide encoding molecule.

[0008] In one embodiment, the method includes providing a separation medium, wherein the separation medium contains at least one target molecule; introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules into the separation medium, wherein the at least two different oligonucleotide encoding molecules include a coding portion operably linked to at least one encoded portion; forming at least two different resolved oligonucleotide encoding molecules by separating the at least two different oligonucleotide encoding molecules into at least two separate locations in the separation medium; harvesting at least one resolved oligonucleotide encoding molecule from the at least two different resolved oligonucleotide encoding molecules by segmenting at least one of the at least two separate locations from the separation medium to form at least one resolved fragment; treating at least one resolved oligonucleotide encoding molecule to allow PCR; amplifying at least one encoded portion of at least one resolved oligonucleotide encoding molecule by PCR on the coding portion of at least one resolved oligonucleotide encoding molecule; and collecting target activity data for at least one resolved oligonucleotide encoding molecule by associating at least one position with a feature of at least one encoded portion of at least one resolved oligonucleotide encoding molecule.

[0009] In one embodiment of the method, at least one target molecule includes at least one of cells, oligonucleotides, proteins, enzymes, ribosomes and nanodiscs. In one embodiment of the method, the separation medium contains at least one of particles, polymers and separation surfaces, and at least one target molecule is connected to at least one of the separation medium, particles, polymers and separation surfaces. In one embodiment of the method, the particles include polymer particles or metal colloids. In one embodiment of the method, the polymer has a molecular weight of 10% or more of the minimum weight target molecule of at least one target molecule. In one embodiment, the method includes separating at least two different oligonucleotide encoding molecules based on at least one target activity between at least one target molecule and the encoding portion of at least two different oligonucleotide encoding molecules. In one embodiment of the method, at least one target activity includes chemical modification of the encoding portion of at least one oligonucleotide encoding molecule by at least one target molecule. In one embodiment of the method, the oligonucleotide contains at least two coding regions, at least one encoded portion contains at least two position building blocks, and each position building block of at least one encoded portion is identified by 1 to 5 coding regions of the oligonucleotide. In one embodiment of the method, the separation medium contains porous gel and a buffer system.

[0010] In one embodiment of the method, at least two different oligonucleotide encoding molecules have a structure according to formula (I),

[0011] (I)G-L-B

[0012] in

[0013] G comprises an oligonucleotide comprising at least two coding regions;

[0014] B is an encoded portion containing at least two position building units;

[0015] L is a linker that operably connects G to B; and

[0016] Each position building block in B is identified according to the position of 1 to 5 coding regions of G.

[0017] In one embodiment of the method, at least two different oligonucleotide encoding molecules have a structure according to formula (II),

[0018] (II)[(B1) M —L1] O —G—[(L2—(B2) K ] P

[0019] in

[0020] G comprises an oligonucleotide comprising at least two coding regions;

[0021] B1 is a position building unit, and M represents an integer from 1 to 20;

[0022] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0023] L1 is a linker that operably connects B1 to G;

[0024] L2 is a linker that operably connects B2 to G;

[0025] O is zero or 1;

[0026] P is zero or 1;

[0027] The condition is that at least one of O and P is 1; and

[0028] Therein, the position building blocks B1 at position M and / or B2 at position K are each identified by 1 to 5 coding regions of G.

[0029] In one embodiment of the method, at least two different oligonucleotide encoding molecules have a structure according to formula (III),

[0030] (III)[(B1) M —L1] O —G'—[(L2—(B2) K ] P

[0031] in

[0032] G' comprises an oligonucleotide, G' comprises at least two coding regions and at least one hairpin region (haripin);

[0033] B1 is a position building unit, and M represents an integer from 1 to 20;

[0034] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and M is the same as or different from K;

[0035] L1 is a linker that operably connects B1 to G';

[0036] L2 is a linker that operably connects B2 to G';

[0037] O is an integer from 0 to 5;

[0038] P is an integer from 0 to 5;

[0039] Provided that at least one of O and P is an integer from 1 to 5; and

[0040] Therein the position building blocks B1 at position M and / or B2 at position K are each identified by 1 to 5 coding regions of G'.

[0041] In one embodiment, the method further comprises separating at least two different oligonucleotide encoding molecules into at least two separate positions in the separation medium by applying a first separation treatment throughout the separation medium in a first direction, wherein the first separation treatment comprises a first voltage scheme and a first duration. In one embodiment, the method further comprises harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one position from the separation medium in a first segmentation direction substantially perpendicular to the first direction to form at least one resolved fragment. In one embodiment, the method further comprises separating at least two different oligonucleotide encoding molecules into at least two separate positions of the separation medium by applying a second separation treatment throughout the separation medium in a second direction, wherein the second direction is substantially perpendicular to the first direction, wherein the second separation treatment comprises a second voltage scheme and a second duration. In one embodiment, the method further comprises harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one position from the separation medium in a second segmentation direction substantially perpendicular to the first segmentation direction to form at least one resolved fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The foregoing summary and the following detailed description of the embodiments will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustration, some embodiments are shown in the drawings, which may be preferred. It should be understood that the embodiments depicted are not limited to the precise details shown.

[0043] Figure 1 A flow chart depicting an embodiment of the method disclosed herein.

[0044] Figure 2 is a schematic diagram of an embodiment of the method disclosed herein.

[0045] Figure 3 is a schematic diagram of an embodiment of a method for molding an electrophoresis channel for target activity separation.

[0046] Figure 4 Schematic diagram of an embodiment of a method for performing two-dimensional electrophoresis using two different separation media.

[0047] Figure 5 A chemical representation of the synthetic scheme for fluorescently labeled oligonucleotide encoding molecules.

[0048] Fig. 6A The chemical structure of the positive control compound is shown.

[0049] Figure 6B The chemical structure of the positive control compound is shown.

[0050] Figure 6C The chemical structure of the positive control compound is shown.

[0051] Fig.6D The chemical structure of the positive control compound is shown.

[0052] Fig. 6E The chemical structure of the positive control compound is shown.

[0053] Fig. 6F The chemical structure of the positive control compound is shown.

[0054] Figure 6G The chemical structure of the positive control compound is shown.

[0055] Figure 6H The chemical structure of the positive control compound is shown.

[0056] Fig.6I The chemical structure of the positive control compound is shown.

[0057] Figure 6J The chemical structure of the positive control compound is shown.

[0058] Figure 6K The chemical structure of the positive control compound is shown.

[0059] Figure 6L The chemical structure of the positive control compound is shown.

[0060] Figure 6M The chemical structure of the positive control compound is shown.

[0061] Figure 6N The chemical structure of the positive control compound is shown.

[0062] Fig.6O The chemical structure of the positive control compound is shown.

[0063] Figure 6P The chemical structure of the positive control compound is shown.

[0064] Fig. 7A Contains polarized fluorescence graphs of compounds based on concentration, wherein 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732 correspond to Fig. 6A ,6B , 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, 6K, 6L, 6M, 6N, 6O, and 6P are positive control compounds.

[0065] Figure 7B Contains polarized fluorescence plots of compounds based on concentration.

[0066] Fig. 8A is the chromatogram of the control compound separated by the target activity separation scheme.

[0067] Figure 8B is the chromatogram of the control compound separated by the target activity separation scheme.

[0068] Figure 8C is the chromatogram of the control compound separated by the target activity separation scheme.

[0069] Fig.8D is the chromatogram of the control compound separated by the target activity separation scheme.

[0070] Fig. 9 is a chromatogram of a mixture of reference compounds separated by the target activity separation scheme.

[0071] Fig.10 is a chromatogram of a mixture of reference compounds separated by the target activity separation scheme.

[0072] Fig.11A is a chromatogram of a mixture of reference compounds separated by the target activity separation scheme.

[0073] Fig. 11B is a chromatogram of a mixture of reference compounds separated by the target activity separation scheme.

[0074] Fig.12 is a schematic diagram of a computer system for implementing embodiments of the systems and methods disclosed herein.

[0075] Detailed description

[0076] All measurements are in standard metric units unless otherwise noted.

[0077] Unless otherwise noted, the prefix "u" when used directly as a unit measurement means "micro" and is often abbreviated to "μ." For example, "uL" stands for "microliter" or "μL."

[0078] Unless otherwise stated, all instances of the words "a," "an," or "the" may refer to one or more than one of the word they modify.

[0079] Unless otherwise specified, the phrase "at least one of..." refers to one or more than one or any combination of more than one of the objects. For example, "at least one of H1, H2, and H3" refers to H1, H2, or H3 or any combination thereof.

[0080] Unless otherwise specified, the term "about" refers to ±10% of the non-percent number being described, rounded to the nearest integer. For example, approximately 100 mm may include 90 to 110 mm. Unless otherwise specified, the term "about" refers to ±5% of a percentage number. For example, approximately 20% may include 15% to 25%. When the term "about" is discussed in terms of a range, the term refers to a suitable amount that is less than the lower limit and greater than the upper limit. For example, approximately 100 to approximately 200 mm may include 90 to 220 mm.

[0081] Unless otherwise indicated, the terms "hybridize," "hybridizing," "hybridized," and "hybridization" include Watson-Crick base pairing, which includes guanine-cytosine and adenine-thymine (GC and AT) pairing for DNA and guanine-cytosine and adenine-uracil (GC and AU) pairing for RNA. Often, these terms are used to refer to the nucleotide strand that selectively recognizes the complementary strand of nucleotides, called the anticodon or anti-coding region.

[0082] The phrases "selectively hybridizing," "selective hybridization," "selectively sorting," and "selective recognition" refer to a selectivity of 5:1 to 100:1 or more for complementary oligonucleotide strands relative to non-complementary oligonucleotide strands.

[0083] The term "oligonucleotide encoding molecule" refers to a molecule of the present disclosure comprising an oligonucleotide and at least one encoded moiety.

[0084] The term "encoding portion" refers to a portion of an oligonucleotide encoding molecule that contains an oligonucleotide, wherein the oligonucleotide encodes and can identify the encoded portion of the oligonucleotide encoding molecule.

[0085] The term "encoded portion" refers to one or more parts of an oligonucleotide encoding molecule, which contains the structure of a building block, such as position building blocks B1 and B2, and they are encoded and can be identified by the encoding portion of the oligonucleotide encoding molecule. For example, the term "encoded portion" does not include such as a joint, even if these structures can be added as a part of the process of the encoded portion of the synthesis, because the joint is not encoded by the encoding portion of the oligonucleotide encoding molecule. As a second example, the term "encoded portion" and "encoded portion" will not include the molecular structure introduced after the encoding process, such as a fluorescent side chain.

[0086] The phrase "total number of positional building blocks" refers to the total number of building blocks in the encoded portion. The meaning of the term "building block" may vary depending on the context. The term "building block" generally refers to a chemical change that is encoded in the encoding portion and is used as the encoded portion. The first example of a building block is a chemical subunit that can react with a joint or another building block and combine with the joint or another building block to form a part of the encoded portion. As a second example, a building block can be a chemical change that includes the removal of a chemical portion. Specific examples thereof include, but are not limited to, the hydrolysis of esters, or the deprotection of amines or aldehydes or alcohols. The third example includes building blocks that represent chemical changes made to a joint or another building block, which changes the reactivity of the joint or building block. Specific examples include, but are not limited to, oxidation of alcohols to aldehydes or ketones, reduction of aldehydes or ketones to alcohols, reduction of nitro groups to amines, reduction of azides to amines, or oxidation of amines to nitro groups or azides.

[0087] The term "identified" and "identify" refers to the correlation between the structure and / or sequence of the building blocks of the coded portion of the coding region or a combination of coding regions and the coded portion of the oligonucleotide encoding molecule. Generally speaking, this correlation of the coding region sequence can be combined with the knowledge of the synthesis step for building the coded portion to allow inference or identification of the sequence, structure and / or predicted structure of the coded portion, even if and when the sequence is obtained indirectly from a copy of the PCR produced by the coding portion of the oligonucleotide encoding molecule.

[0088] The terms "first", "second", etc. are understood to be terms that only specify or distinguish which object is being referred to and are generally based on the order of which object is encountered first. For example, the "first" array is the array that is used first, and the first coding region is the first coding region that can be fixed on the first array. Unless otherwise specified, the terms "first", "second", etc. do not refer to positions within a molecule. For example, it should be understood that the first coding region and the second coding region may or may not be continuous and may or may not be close to each other within the coding portion.

[0089] In this disclosure, a hyphen or dash in a molecular formula indicates that the parts of the molecular formula are directly linked to each other by covalent bonding or hybridization.

[0090] Unless otherwise indicated, all ranges of nucleotides, integer values, and percentages include all intermediate integers as well as endpoints. For example, a range of 5 to 10 nucleotides will be understood to include 5, 6, 7, 8, 9, and 10 nucleotides.

[0091] In certain embodiments, the disclosure relates to oligonucleotide encoding molecules (OEM) containing at least one oligonucleotide portion as a coding portion and at least one encoded portion, wherein the oligonucleotide portion uses combinatorial chemistry to guide or encode the synthesis of at least one coding portion. In certain embodiments, the oligonucleotide portion of the oligonucleotide encoding molecule can identify or promote the deduction of at least one encoded portion of the oligonucleotide encoding molecule. In certain embodiments, the oligonucleotide encoding molecule of the present disclosure contains at least one oligonucleotide or an oligonucleotide portion containing at least two coding regions, wherein the combination of at least two coding regions corresponds to and can be used to identify or derive the sequence of the building block in the encoded portion or the sequence of the structure of the encoded portion. In certain embodiments, at least one oligonucleotide or oligonucleotide portion can be amplified by polymerase chain reaction (PCR) to produce a copy of at least one oligonucleotide or oligonucleotide portion. In one embodiment, the original oligonucleotide or oligonucleotide portion or its copy can be sequenced to determine the characteristics of the combination of at least two coding regions of the oligonucleotide encoding molecule. In certain embodiments, the characteristics of the combination of at least two coding regions can be related to a series of combinatorial chemical steps for synthesizing the encoded portion of the oligonucleotide encoding molecule. In certain embodiments, a series of combinatorial chemistry steps used to synthesize the encoded portion can identify or allow for the derivation of an oligonucleotide encoding the encoded portion of the molecule.

[0092] Methods for synthesizing oligonucleotide-encoded molecular libraries have been and continue to be the focus of intensive academic and industrial research due to their ability to systematically evaluate millions to billions of molecules for intermolecular properties, such as target activity, against a target molecule. For example, if the target molecule is a key enzyme in a cancer pathway, then researchers can determine whether the encoded portion of one or more of the millions to billions of oligonucleotide-encoded molecules can bind to the enzyme in a way that increases or decreases the rate of its catalytic reaction. By analogy, this is like determining the combination of a digital lock by using a device that can electronically try millions of combinations in a short period of time until the right combination is found. However, the digital lock is designed to be unlocked by the correct digital sequence, and the biological target may not have the correct structural combination, but it is hoped that one or more molecules can be discovered that work well enough to be commercialized as an effective therapy. To address this challenge, oligonucleotide-encoded molecular libraries can use a kind of guided evolution to get closer and closer to molecules that may have the desired binding affinity. For example, if an oligonucleotide-encoded molecule reacts weakly with a target molecule, the next oligonucleotide-encoded molecular library can be synthesized to explore structural changes in the encoded portion of the most promising candidate, hoping to discover an encoded portion with better binding properties. This guided evolution and evaluation can proceed until a valid solution is found, or a dead end is reached, such that the next molecule is selected as the starting point for the guided evolution study.

[0093] However, although this research area is expected to find molecules with useful properties, the vast majority of research has been devoted to the development of effective methods for synthesizing oligonucleotide encoded molecule libraries. The next generation method of sequencing and identifying the encoded portion of oligonucleotide encoded molecules from the coding portion or its polymerase chain reaction (PCR) copy has also achieved considerable development. Also paid considerable effort to apply statistical techniques to obtain larger dimensions from sequencing data. How to test the desired properties of oligonucleotide encoded molecules on the target molecule is less progress.

[0094] A traditional method for testing whether a library of oligonucleotide-encoded molecules reacts with a target molecule is a "large-scale exposure" method, which is referred to as "panning" in some cases. The large-scale exposure method simply exposes the target molecule to a library of oligonucleotide-encoded molecules or a portion thereof in a solvent or medium. Typically, the target molecule is fixed and the oligonucleotide-encoded molecule binds to the target molecule. After exposure for a period of time, the solvent or medium is removed, leaving the attached target molecule of the strongly bound oligonucleotide-encoded molecule or associating with the target molecule. These strongly bound oligonucleotide-encoded molecules can be identified by: using PCR to make a copy of the coding portion, and then sequencing the copy or original to decode and identify the structure of the strongly bound coding portion. This method can find which members or special oligonucleotide-encoded molecules strongly bind or associate with the target molecule. However, this large-scale exposure method is unreliable, and the binder (binder) suite (suit) determined in the replicate experiment may vary greatly. Typically, only the strongest binder is captured reproducibly each time, even though there may be many molecules that are only medium or weak binders, but their structure can provide valuable insights for associating chemical structure with biological activity. In addition, the method can provide false positives, where the member that remains fixed in the test chamber binds to some part of the test environment, such as the chamber itself, or the tethered medium, or another oligonucleotide encoding molecule. The large-scale exposure method also provides false negatives, because molecules that weakly, moderately, or even strongly bind to the target molecule can bind, release, and then be washed away when the solvent is removed. The molecules that bind, release, and are washed away can never be recovered or identified in the sequencing data, so this false negative caused by the test loss (assayattriton) makes it impossible to distinguish true binders from non-binders. The second source of false negatives is the presence of many false positives. Typically, false positives provide a stronger signal than true binders in the sequencing data. In this case, one cannot distinguish the true binders from the false positives, and the true binders are lost in the noise. In fact, this large-scale exposure method does not provide data measuring the target affinity of the oligonucleotide encoding molecule to the target molecule. The data generated by this standard method is binary: presence during PCR and sequencing means binding; and absence during PCR means unbound. From a processing point of view, the large-scale exposure method may be effective, but from a data acquisition point of view, it is inefficient and limited. Since data acquisition is the main goal of high-throughput screening of oligonucleotide-encoded molecular libraries, large-scale exposure methods have become a bottleneck in the drug discovery process.

[0095] Another traditional method for testing whether the oligonucleotide encoding molecule library binds to the target molecule is the "mass exposure followed by electrophoresis" method, which is called "gel shift assay" in some cases. In this method, the large-scale exposure followed by electrophoresis method simply exposes the target molecule to the oligonucleotide encoding molecule library in a solvent or medium in a manner similar to the "mass exposure" method discussed previously, except that the target molecule is not bound. In contrast, the mixture of the oligonucleotide encoding molecule library that binds the target molecule, the unbound target molecule, and the unbound oligonucleotide encoding molecule is purified by subjecting the mixture to traditional electrophoresis. Traditional electrophoresis separates the oligonucleotide encoding molecules that bind to the target molecule according to the difference between molecular size and charge, which can separate the target molecule bound to the oligonucleotide encoding molecule from the unbound target molecule and the unbound oligonucleotide encoding molecule. This method of large-scale exposure followed by electrophoresis can have the benefit of separating those oligonucleotide encoding molecules that bind to the target molecule from those oligonucleotide encoding molecules and the target molecule that remains unbound. However, this traditional technique suffers from the same false negatives and provides the same binary data: bound or unbound.

[0096] The present disclosure relates to methods for separating oligonucleotide-encoded molecules by applying a "target active electrophoresis". As a general overview of the methods disclosed herein, reference is made to Figure 1 and Figure 2The method includes providing a separation medium, wherein the separation medium contains at least one target molecule, 102, 202; introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules into the separation medium, wherein the at least two different oligonucleotide encoding molecules include a coding portion (e.g., "CBA") operably linked (e.g., "L") to at least one encoded portion ("star"), 104, 204; forming at least two different resolved oligonucleotide encoding molecules by separating the at least two different oligonucleotide encoding molecules into at least two separate locations in the separation medium, 106, 206 (relative target activity described by the size of the lightning symbol); and measuring the migration distance 108, 208 (e.g., D) by splitting at least one of the at least two separate locations from the separation medium to form at least one resolved fragment. 1 or D2 depicted), to harvest at least one resolved oligonucleotide encoding molecule from at least two different resolved oligonucleotide encoding molecules; treating at least one resolved oligonucleotide encoding molecule for PCR, 110, 210; amplifying at least one coding portion of at least one resolved oligonucleotide encoding molecule by PCR on the coding portion of at least one resolved oligonucleotide encoding molecule, 112, 212; sequencing the coding portion of the resolved oligonucleotide encoding molecule or a PCR copy thereof, and identifying at least one encoded portion of at least one resolved oligonucleotide encoding molecule, 114, 214; collecting target activity data of at least one resolved oligonucleotide encoding molecule by associating at least one position with a feature of at least one encoded portion of at least one resolved oligonucleotide encoding molecule, 116, 216.

[0097] Generally speaking, the method comprises introducing the mixture of oligonucleotide encoding molecules into the separation medium containing target molecules, and using electrophoresis, oligonucleotide encoding molecules are migrated through the separation medium containing target molecules, so that oligonucleotide encoding molecules are separated at least in part based on the activity between the encoded part of target molecules and oligonucleotide encoding molecules. Then, in one embodiment, the method may include the separation medium containing separated or resolved oligonucleotide encoding molecules is segmented, and the migration distance of different fragments from starting point or sample well is measured. Then, in one embodiment, the method may include harvesting oligonucleotide encoding molecules with the pcr amplification of the encoding part of oligonucleotide encoding molecules by processing separation medium. In one embodiment, the method may include carrying out pcr amplification and sequencing of the encoding part of oligonucleotide encoding molecules, then the associated sequence data is sequenced to identify the encoded part of oligonucleotide encoding molecules, and the encoding part is sequenced, and the encoded part of oligonucleotide encoding molecules is identified or derived. In one embodiment, the method may include collecting target activity data by associating the feature of the encoded part with its migration distance or position in the separation medium.

[0098] In more detail, in one embodiment, the method can fix or reduce the mobility of the target molecule in the medium. In one embodiment, the oligonucleotide encoding molecule library can be introduced into the separation medium at the sample well. Then the oligonucleotide encoding molecule library can be subjected to electrophoresis, so that the oligonucleotide encoding molecule library is migrated through the medium to contact with the fixed target molecule. Further, in one embodiment, the oligonucleotide encoding molecule library can be separated or parsed based on their activity with the target molecule in part. In one embodiment, the oligonucleotide encoding molecule with high activity to the target molecule will slow down its migration through the separation medium, and the oligonucleotide encoding molecule with low activity to the target molecule will slow down its migration. In one embodiment, once enough separations are achieved, the part of the sample mixture containing the oligonucleotide encoding molecule can be recovered by dividing the medium into several parts. In one embodiment, these parts can be separated by dissolving them in a solvent or soaking them in a solvent to allow the oligonucleotide encoding molecule in the part to enter the solvent. In one embodiment, once a part of the oligonucleotide encoding molecule is recovered, the encoded part of the oligonucleotide encoding molecule can be determined by performing PCR to form a copy of the encoded part of the oligonucleotide encoding molecule. In one embodiment, copies of the encoded portion of an oligonucleotide encoding molecule can be sequenced and the sequence data can be used to identify the oligonucleotide encoding molecule and measure the migration distance of the oligonucleotide encoding molecule in a separation medium.

[0099] In one embodiment, a benefit of this method can be to avoid or reduce false negatives, because even strongly bound or reactive oligonucleotide-encoded molecules can be restored by applying a stronger voltage, making it unnecessary to infer based on the disappearance of the molecule in the detection. In one embodiment, the benefit of this method can be that the measurement of the migration distance of the oligonucleotide-encoded molecule can provide qualitative and / or quantitative data of the affinity of the oligonucleotide-encoded molecule to the target molecule. In contrast, traditional methods can only provide binary data for each molecule: binding or non-binding. In one embodiment, the benefits of the currently disclosed method can include measuring different types of interactions. For example, traditional methods can only measure probe-target affinity. In contrast, the method disclosed herein can provide data on the chemical reactivity of the encoded portion of the oligonucleotide-encoded molecule, because the chemical reactivity of the target molecule that reacts (e.g., catalytic reaction) with the encoded portion tends to slow down the migration rate through the separation medium.

[0100] In one embodiment, the currently disclosed method can greatly increase the number of real binders captured and identified. First, since there is no "washing" step, real binders are less likely to be lost due to experimental loss. The washing step in the large-scale exposure / panning method uses liquid flow and is intended to move molecules that cannot bind to the target away from the target. However, the real effect is to move unbound molecules - that is, washing will also move (a) molecules that cannot bind and (b) molecules that are only temporarily unbound. In contrast, the method disclosed herein uses electrophoresis to move molecules that are not bound to the target, but instead moves them from one place with a target to another place with a target; this provides a greater opportunity for temporarily unbound molecules to rebind. Because this process is repeated many times along the migration path, there are far fewer compounds lost as false negatives due to experimental loss. Secondly, because the voltage in the system applies a very strong and continuous force to molecules that bind in a transient, non-specific manner, these molecules are removed more thoroughly than in the washing process. Because they are removed more completely, these compounds produce smaller signals in the sequencing data and are therefore less likely to drown out the signals of real binders. The net effect is a large increase in the signal-to-noise ratio and a large increase in the number of compounds identified as true binders.

[0101] In one embodiment, the method includes providing a separation medium, wherein the separation medium contains at least one target molecule. In one embodiment, the separation medium is generally unrestricted, as long as the medium allows the electrophoresis of the oligonucleotide encoding molecule. Suitable separation medium includes porous gel and a buffer system suitable for electrophoresis. Suitable porous gel may include agarose, polyacrylamide, various hydrogels and starch. Suitable buffer systems may include Tris / acetate / EDTA (TAE), Tris / borate / EDTA (TBE), Tris / borate (TB) and lithium / borate (LB), wherein EDTA represents ethylenediaminetetraacetic acid, and Tris represents tris (hydroxymethyl) aminomethane. The gelling material of various concentrations in the selected buffer system may be used to control the porosity of the gel.

[0102] In one embodiment, the method comprises providing a separation medium comprising at least one target molecule.

[0103] In one embodiment, the target molecule can be fixed, or the mobility of the target molecule can be reduced by binding or tethering the target molecule to at least one of a separation medium, a particle, a polymer, and a separation surface. In one embodiment, the target molecule can be bound or tethered to at least one of a particle, a polymer, and a separation surface before, during, or after the target contacts the separation medium. In one embodiment, the target molecule is bound or tethered to the particle before adding a separation medium such as agarose gel. In general, any suitable method for binding a molecule to a surface or other molecules can be used, as long as the binding is stable during electrophoresis.

[0104] In one embodiment, the target molecule, such as a protein or protein complex, can be bound to an anchor including a separation medium, a particle, a polymer, and a separation surface using various binding methods known in the art. Suitable binding methods include amide bond cross-linking, sulfonamide or sulfone formation, weakly reactive electrophilic interactions, polymerization, disulfide formation, ester formation, click reactions (such as azide alkyne reactions with copper), Diels-Alder cycloadditions and cross-metathesis, calcium alginate immobilization by matrix capture, and the like.

[0105] It is known that fixing molecules such as target molecules on solid surfaces can cause or at least risk the molecules to deform, which can hide the activity of natural molecules. Methods to avoid or reduce target molecule deformation can be advantageous because they allow the natural activity of target molecules to be measured. In one embodiment, the target molecule is connected, combined, strongly associated or tethered to a polymer or oligomer (polymer and / or oligomer different from a separation medium), wherein the polymer or oligomer has a molecular weight of 10% or more, including 20% ​​to 5000% of the target molecule's lowest molecular weight. In one embodiment, the benefit of the target molecule being tethered to a polymer or oligomer can be that the combination of oligonucleotide encoding molecules, target molecules and polymers or oligomers can migrate through the separation medium at different rates, thereby allowing further separation with other molecules, while eliminating or reducing the risk of target molecule deformation. In one embodiment, the benefit of the target molecule being tethered to a polymer or oligomer can be that the oligonucleotide encoding molecules, target molecules and polymers or oligomers can be removed from the separation medium into, for example, a solvent or buffer before PCR and sequencing the coding portion to identify the encoded portion.

[0106] In one embodiment, the method includes a separation medium, wherein the separation medium contains a target molecule bound or tethered to a particle. In one embodiment, the particle can be a composition labeled with an appropriate anti-target tag. In one embodiment, the particle can be a solid particle, an amorphous particle, a porous particle, a polymeric particle, a metal colloid, a mixture of materials, or a monomer electrophoresis medium. Suitable particles may include ion exchange resins, silica particles, polystyrene, agarose beads, biotin-labeled agarose, Beads, Resin beads, dendrimers (polyethylene glycol, polystyrene, etc.), (magnetic particles); and calcium alginate immobilized by matrix capture. In one embodiment, one benefit of binding or tethering a target molecule to a particle may be that the target molecule is immobilized or its migration during electrophoresis is slowed relative to unbound target molecules.

[0107] In one embodiment, the benefit of attaching or tethering the target molecule to a particle may be that the particle is immobilized in a separation medium. In one embodiment, the target molecule is attached or immobilized on the surface of a gel electrophoresis plate, wherein the gel electrophoresis plate is the surface on which the gel is formed. In one embodiment, the benefit of attaching or tethering the target molecule to a particle or surface may be to limit or prevent the migration of the target molecule, thereby removing the mobility of the target molecule as the basis for separation.

[0108] Methods that can selectively bind target molecules to separation media, particles, polymers, or separation surfaces can be advantageous because they allow the use of different binding mechanisms, which can remove the choice of binding mechanism from consideration for multiple experiments. In one embodiment, a conjugate pair reaction selectively binds a labeled target molecule to a separation media, particle, polymer, or separation surface. Suitable conjugate pair reactions include His tags (wherein His is histidine) (in integers between 6-10) with particles containing or displaying nickel NTA or anti-His antibodies on their surfaces; biotin tags with particles containing streptavidin, avidin, or anti-biotin antibodies; streptavidin binding peptides with particles containing streptavidin or avidin; Halo-Tags with particles displaying Halo-Tag proteins; FLAG tags with particles containing or displaying anti-FLAG antibodies; calmodulin binding proteins with particles containing or displaying calmodulin glutathione S-transferase with particles containing glutathione; cellulose binding domain (CBP) with cellulose particles or separation media; native protein with particles containing or displaying anti-protein antibodies or covalently tethered to particles via common linking chemistry (e.g., N-hydroxysuccinimide or carbodiimide chemistry) via surface lysine moieties that react with carboxyl groups on the particle surface; streptavidin protein with particles containing or displaying biotin or anti-streptavidin antibodies; and oligonucleotide-tagged proteins with particles containing or displaying complementary oligonucleotides.

[0109] In one embodiment, the method includes a target molecule in a separation medium. In one embodiment, the method may include adding or mixing the target molecule into a separation medium before introducing a sample or mixture of oligonucleotide-encoded molecules into the separation medium. In one embodiment, the method may include fixing or binding the target molecule to a separation medium or a surface in contact with or contained by the separation medium. In one embodiment, the target molecule may be a cell, including a stem cell or a cancer cell; an oligonucleotide, including DNA (deoxyribonucleic acid) and RNA (ribonucleic acid); a target of a natural cell lysate, an overexpressed cell lysate; a natural protein, a mutant protein, a peptide, an enzyme, including but not limited to cytochromes, kinases, glutaminases, phosphorylases, ribosomes, liposomes, synthetic molecules, and nanodiscs, including mixtures of each, some, or all. Suitable synthetic molecules may include drugs and pollutants. In one embodiment, a nanodisc may include a lipid bilayer of phospholipids, whose hydrophobic edges are masked by two amphipathic proteins. Such nanodiscs are typically used to study membrane proteins. In one embodiment, the target molecule is attached to a particle, including a nanotube, a polymer, a nanoparticle, or a colloid.

[0110] In one embodiment, the target molecule can be evenly or substantially evenly distributed in the separation medium along the migration axis, wherein the migration axis can be the voltage direction across the separation medium. In one embodiment, the target can be distributed with a concentration gradient that increases or decreases relative to the migration direction, to increase or reduce the separation of the oligonucleotide encoding molecule. In one embodiment, the target molecule can be tethered to a particle or polymer, and then the tethered target can be mixed into the separation medium before the medium solidifies or gels, and the mixture can be centrifuged to provide the gradient concentration of the target tethered in the separation medium. A benefit of tethering the target to a polymer or a particle is to select a polymer and a particle that the target is distributed in the separation medium according to the profile of the design. For example, a group of homogeneous target molecules can be tethered to one of two particles of different sizes, so that when the separation medium solidifies or gels, two groups or bands of target molecules are formed.

[0111] In one embodiment, the target molecule can be mixed into the separation medium before the medium solidifies or gels, and the mixture can be centrifuged to provide a target molecule gradient concentration in the separation medium. Suitable centrifugation methods may include differential centrifugation, rate zonal centrifugation, and isopycnic centrifugation. In one embodiment, the concentration gradient of the target material in the separation medium can be provided by size exclusion fractionation, material electrophoresis, magnetic separation of magnetic targets, based on timed retention separated from other chromatographic separation techniques, or any suitable method for manipulating the target in a liquid medium.

[0112] In one embodiment, the method may include mixing a certain amount of target molecule with a certain amount of liquid separation medium to provide a target molecule concentration in the separation medium. In one embodiment, the concentration of the target molecule in the separation medium can be in the range of about 500 μg / mL to about 5 mg / mL. In one embodiment, the method may include contacting, mixing or combining the target molecule with a particle, polymer or oligomer, and then adding the mixture of the target molecule and the particle, polymer or oligomer to the separation medium. In one embodiment, the method may include adding the target molecule and the particle, polymer or oligomer to the separation medium simultaneously or in any order.

[0113] In one embodiment, the method includes providing a separation medium, wherein the separation medium contains at least one target molecule and at least one sample area or sample well. In one embodiment, the method includes adding, pouring and / or molding the separation medium onto a flat surface of an electrophoresis plate to provide a substantially flat, continuous separation medium. Such a flat, continuous separation medium is shown in Figure 4 ,402. Reference Figure 3 , the separation medium can be molded or formed as Figure 3 Lanes of the separation medium shown. In one embodiment, the method includes providing a mold with a lane ridge on the top surface (cast bearing lane ridge) 302; pouring a suitable polymer, such as polydimethylsiloxane (PDMS), onto the top surface of the mold with the lane ridge and crosslinking or gelling it 304; directing the molded polymer so that the lane channel faces upward 306; optionally, closing portions 308, 310 of the mold; and filling the lane channel with a separation medium to form a separation medium 312 shaped as a separation lane. In one embodiment, the material of the closed mold portion can be removed to form a sample well 312. In one embodiment, after the separation medium solidifies or gels, the sample well can be cut into separate parts. It has been found that higher resolution of oligonucleotide encoding molecules with different activities from the target can be obtained by target activity electrophoresis than by separation using a continuous separation medium or capillary electrophoresis. Do not wish to be limited by theory, it is believed that the advantage of using capillary electrophoresis to separate is that target is not labeled, but when the oligonucleotide encoding molecules obtain larger effective molecular weight than free ones when combined with the target, complete separation, thereby migrate in different ways.Yet, achievable separation degree is limited to the different mobilities of mobile, unconjugated oligonucleotide encoding molecules and mobile, combined oligonucleotide encoding molecules.Target activity electrophoresis can obtain higher resolution, because the oligonucleotide encoding molecules in conjunction with the fixed target have obtained effective infinite molecular weight, to which they can not move at all when combined, and the degree that unconjugated oligonucleotide encoding molecules can still move freely with the maximum rate of the system.

[0114] In one embodiment, the method includes forming at least two different resolved oligonucleotide encoding molecules by separating at least two different oligonucleotide encoding molecules into at least two separate locations in a separation medium, wherein the separation medium is molded or shaped into a separation lane, wherein the separation lane has a radius of width and a radius of depth, and the width radius R1 and the depth radius R2 can be the same or different, and can increase, decrease or remain consistent along the length of the separation lane in the migration direction.

[0115] In one embodiment, the method may include applying a first separation process throughout a separation medium in a first direction, separating at least two different oligonucleotide encoding molecules into at least two separate positions in a separation medium, wherein the first separation process includes a first voltage scheme and a first duration. In one embodiment, the method may include harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one position from a separation medium in a first segmentation direction substantially perpendicular to the first direction to form at least one resolved fragment. In the context of direction or axis, the term "substantially" refers to within 30 degrees. It is understood that the more aligned with the indicated direction, the better the effect. If the first separation process is sufficient, further purification or separation method may not be required. However, if the first separation process does not provide the required resolution, the subsequent second time or continuous processing can be applied. For example, after applying the first treatment in one direction, the second treatment can then be applied by applying electrophoresis conditions in the second direction.

[0116] In one embodiment, the method comprises applying the second separation process throughout separation medium by at least two different oligonucleotide encoding molecules being separated to at least two separated positions of separation medium, wherein the second direction is substantially perpendicular to the first direction, wherein the second separation process comprises the second voltage scheme and the second duration.In one embodiment, the method comprises the oligonucleotide encoding molecules of at least one resolution being gathered in the crops by cutting at least one position from separation medium to form at least one resolved fragment on the second segmentation direction substantially perpendicular to the first segmentation direction.In one embodiment of the method, when the oligonucleotide encoding molecules remain in the same separation medium, the first and second separation processes are applied.In one embodiment, the benefit of applying two-dimensional electrophoresis to the mixture of oligonucleotide encoding molecules can be based on applying different second voltage parameters, comprising different voltages, different rates of change or ramp voltages or with respect to the pulse voltage of the first voltage parameter applied, improving the separation of the mixture of oligonucleotide encoding molecules.This embodiment is consistent with two-dimensional electrophoresis known in the art.

[0117] refer to Figure 4 In one embodiment, the method includes providing a first separation medium, wherein the first separation medium contains at least one target molecule, 402; and separating at least two different oligonucleotide encoding molecules into at least two separate locations in the separation medium by applying a first separation treatment throughout the separation medium in a first direction, wherein the first separation treatment includes a first voltage scheme and a first duration, 404; segmenting a portion or plug of the first separation medium from the first separation medium, including along a separation line, lane, or axis, 406; inserting or plugging the plug from the first separation medium into a sample well of a second separation medium, 408, 410; and separating at least two different oligonucleotide encoding molecules into at least two separate locations of a second separation medium by applying a second separation treatment throughout the separation medium in a second direction, 412, wherein the second direction is substantially perpendicular to the first direction, wherein the second separation treatment includes a second voltage scheme and a second duration. In one embodiment, the method includes harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one location from the second separation medium in a second segmentation direction substantially perpendicular to the first segmentation direction to form at least one resolved fragment. In one embodiment, the first and second separation media can be the same or different. In one embodiment, the first separation medium may contain a first target molecule and the second separation medium may contain a second target molecule, wherein the first target molecule may be the same or different from the second target molecule. In one embodiment, the concentrations of the first target molecule and the second target molecule may be the same or different.

[0118] In one embodiment, the separation medium comprises at least one sample area or sample well. In one embodiment, the at least one sample area is separated from the area comprising at least one target molecule, wherein the range of the interval is 1mm to 10cm. In one embodiment, at least one sample area may include one or more holes or holes cut into the separation medium. In one embodiment, the benefit of the sample area may include a place for introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules before electrophoresis or electrophoretic separation. In one embodiment, the method may include measuring the migration distance from the edge of the sample area to the edge of the resolved fragment, wherein the edge of the sample area is the edge in the migration direction.

[0119] In one embodiment, a mixture of at least two different oligonucleotide encoding molecules or a portion thereof can be contacted with a target molecule by subjecting the sample to electrophoresis, causing at least two different oligonucleotide encoding molecules to migrate from a sample region to contact with at least one target molecule. The electrophoretic method is generally not limited as long as the method is capable of contacting at least a portion of the mixture with a target molecule and / or causing at least two different oligonucleotide encoding molecules to migrate through a separation medium. In one embodiment, the method of applying electrophoresis does not result in degradation of the oligonucleotide encoding molecules, the target molecule, or the particles or polymers (if present) tethered to the target molecule.

[0120] In one embodiment, the method comprises applying the first separation treatment throughout the separation medium in the first direction, separating at least two different oligonucleotide encoding molecules to at least two separated positions in the separation medium, wherein the first separation treatment comprises a first voltage scheme and a first duration. In one embodiment, the method comprises applying the second separation treatment throughout the separation medium in the second direction, separating at least two different oligonucleotide encoding molecules to at least two separated positions of the separation medium, wherein the second direction is substantially perpendicular to the first direction, wherein the second separation treatment comprises a second voltage scheme and a second duration. It should be understood that the separation step can be repeated frequently as required by applying other separation treatments to other voltage schemes with other durations and optionally in other directions. In one embodiment of the method, the first separation treatment may include applying about 5V to about 150V, including about 30V to about 140V, including the first voltage scheme of about 50V to about 120V, carrying out about 1 to 50 hours, including about 2 to about 40 hours, including the first duration of about 3 to about 30 hours. In one embodiment of the method, the second separation treatment may include applying a second voltage scheme of about 20V to about 150V, including about 30V to about 140V, including about 50V to about 120V, for about 1 to 50 hours, including about 2 to about 40 hours, including a second duration of about 3 to about 30 hours. In one embodiment, the first treatment scheme and the second treatment scheme may each independently include increasing the applied voltage at a rate from about 1V / hr to about 5V / hr. In one embodiment, the first treatment scheme and the second treatment scheme may each independently include reducing the applied voltage at a rate from about 1V / hr to about 5V / hr. It should be understood that different voltage schemes can be used for separation media of different lengths. Generally, longer lengths require higher voltages to provide shorter separation times. In one embodiment, the method may include placing the separation medium between the anode and the cathode and applying a voltage ranging from about 1V / cm to about 35V / cm throughout the separation medium or gel. In one embodiment, the range of the separation medium may be from about 3cm to about 75cm. In one embodiment, the first treatment scheme and the second treatment scheme may include applying a pulsed current. In one embodiment, the first and second voltage regimes may comprise heating, cooling or maintaining the separation medium at a temperature of about 2°C to about 60°C, including 3°C to about 10°C, including 10°C to about 30°C, including 30°C to about 40°C.

[0121] In one embodiment, the method may include harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one position from the separation medium in a first segmentation direction that is substantially perpendicular to the first direction to form at least one resolved fragment. In one embodiment, the method may include harvesting at least one resolved oligonucleotide encoding molecule by segmenting at least one position from the separation medium in a second segmentation direction that is substantially perpendicular to the first segmentation direction to form at least one resolved fragment. Unless otherwise specified, the phrase "substantially perpendicular direction" as applied to segmentation refers to cutting or slicing at an angle measured from about 70° to about 120° to the direction of migration of the oligonucleotide encoding molecule through the separation medium. The term "segmentation" is generally not limited, as long as the separation medium is divided into several parts. For example, when the separation medium is a gel, then segmentation may include cutting the gel into fragments. In one embodiment, if the separation medium is a gel, then the method may include freezing the gel and segmenting the frozen gel by cutting the frozen gel with a scalpel, a razor or a laser. In addition, when the separation medium is a liquid, then segmentation may include removing an aliquot by pipetting to form resolved liquid fragments. Reference Figure 2 In one embodiment of the method, the migration distance (e.g., D1, D2) of one or more fragments of the separation medium or resolved fragments is measured before, during or after the one or more fragments of the separation medium are severed or cut from the separation medium.

[0122] In one embodiment, before, during or after the separation medium is divided, at least one resolved oligonucleotide encoding molecule can be treated to allow PCR. As long as the separation medium is changed to allow PCR, this step is generally not limited. In one embodiment, if the separation medium is a liquid, the method can omit this step. In one embodiment, removing the portion of the oligonucleotide encoding molecule from the separation fragment can include soaking or wetting the separation fragment of the separation medium in a solvent until at least a portion of the oligonucleotide encoding molecule diffuses from the fragment into the solvent. In one embodiment, the method may include soaking the fragment or resolved fragment in water or a solvent to allow OEM to be discharged from the separation medium. In one embodiment, the method may include, if the separation medium has a melting temperature between about 20°C and about 100°C, heating the fragment or resolved fragment in a buffer solution between about 20°C and about 100°C. In one embodiment, the method may include heating the fragment or resolved fragment to about 20°C to about 100°C and adding an enzyme capable of dissolving the gel. In one embodiment, if the separation medium is agarose, the method can include treating at least one resolved oligonucleotide encoding molecule by adding agarase, including alpha and / or beta agarase, including beta-agarase I (NEB of Ipswich, MA) to allow for PCR.

[0123] In one embodiment, a method can include amplifying at least one encoded portion of at least one oligonucleotide encoding molecule parsed by carrying out PCR to the coding portion of at least one oligonucleotide encoding molecule parsed to form a copy of the coding portion of at least one oligonucleotide encoding molecule parsed. The benefit of the oligonucleotide encoding molecule parsed using PCR amplification can include improving the signal-to-noise ratio of the oligonucleotide encoding molecule parsed interested. The benefit of the oligonucleotide encoding molecule parsed using PCR amplification can include understanding the characteristics of the encoded portion, the coding portion is irreversibly bound to the target molecule, or is difficult to remove from the separation medium due to unforeseen reactions. The PCR program can be adjusted as needed by variations known in the art.

[0124] In one embodiment, the method can include sequencing the coding portion of the oligonucleotide encoding molecule and / or more likely sequencing the coding portion of the PCR copy of the coding portion of the oligonucleotide encoding molecule to identify or derive the sequence, structure or expected structure of the encoded portion of the oligonucleotide encoding molecule. The program of sequencing the PCR copy of the oligonucleotide encoding molecule and the oligonucleotide encoding molecule can be adjusted as needed by changes known in the art, including application of next generation DNA sequencing, large-scale parallel or deep sequencing, which are currently being studied and developed because these methods can save time and money. In one embodiment, the method includes identifying the sequence of a portion of the copy sequence, to identify or associate each coding region or coding region combination of this part of the oligonucleotide encoding molecule, to identify or associate each position building unit of at least one encoded portion. In one embodiment, by sequencing the coding portion of the oligonucleotide encoding molecule or its copy sequence to identify, determine or derive the encoded portion of the oligonucleotide encoding molecule, this can include associating the oligonucleotide sequence in the coding portion with the sequence of the synthesis step for synthesizing the encoded portion.

[0125] In one embodiment, the method can include collecting the target activity data of at least one oligonucleotide encoding molecule analyzed by associating at least one position with the feature of at least one encoded portion of the oligonucleotide encoding molecule analyzed. In one embodiment, the feature of the encoded portion of the oligonucleotide encoding molecule or the oligonucleotide encoding molecule analyzed can be related, matched or associated with the migration distance measured. It is believed that, relative to the oligonucleotide encoding molecule having high activity to the target molecule, the oligonucleotide encoding molecule having low activity to the target molecule will migrate quickly by the separation medium, because those molecules with high activity slow down or are hindered in the reaction process. In addition, without wishing to be bound by theory, it is believed that the oligonucleotide encoding molecule interacting with the target molecule will have k on and k off , where k onis the rate at which the oligonucleotide encoding molecule reacts, interacts or associates with the target molecule, k off It is the rate at which the oligonucleotide encoding molecule dissociates or separates from the target molecule. Generally speaking, it has been observed that the OEM with tighter affinity migrates slower, and the OEM with looser affinity migrates faster. However, it should be understood that target activity is not necessarily a factor affecting electrophoretic migration. For example, electrophoretic rate can be increased based on smaller molecular size and molecules with higher net negative charge. These factors can be offset by introducing appropriate control molecules. Another method for separating target activity from other electrophoretic factors can be to introduce two or more encoded parts for each oligonucleotide encoding molecule. For example, an OEM with an encoded part is expected to slow down its migration by a residence time. Obviously, then the activity of the OEM with two or three encoded parts will be 2 or 3 times larger than the OEM with an encoded part, so that, relative to the OEM with only 1.5 to 3 times of residence time, the migration of the OEM with 2-3 encoded parts will be slowed down. The benefit of the method disclosed herein can be that the use of the OEM with multiple encoded parts can be for the purpose of calculating target activity data, and the target activity is isolated as a factor relative to other factors. One benefit of this approach may include enhancing the signal-to-noise ratio using OEMs with multiple encoded moieties, thereby amplifying differences in their residence times and, therefore, their calculated target reactivities, by introducing multiple encoded moieties per OEM.

[0126] In one embodiment, the method may include measuring the first migration distance of the oligonucleotide encoding molecule from at least one sample area, and the distance of migration is associated with the identification of the encoded portion of the oligonucleotide encoding molecule. In one embodiment, the method may include measuring the second migration distance of the second oligonucleotide encoding molecule from at least one sample area, and the second migration distance is associated with the identification of the encoded portion of the second oligonucleotide encoding molecule. In one embodiment, the method may include calculating the relative or qualitative binding affinity of the first oligonucleotide encoding molecule to the target molecule relative to the second oligonucleotide encoding molecule by dividing the first distance by the second distance.

[0127] In one embodiment, the method may comprise one or more oligonucleotide encoding molecules having a structure according to formula (I),

[0128] (I)G-L-B

[0129] in

[0130] G comprises an oligonucleotide comprising at least two coding regions;

[0131] B is an encoded portion containing at least two building blocks;

[0132] L is a linker that operably connects G to B; and

[0133] Each building block or positional building block in B is identified according to the position of 1 to 5 coding regions of G.

[0134] In one embodiment, the method may include one or more oligonucleotide encoding molecules having a structure according to formula (II),

[0135] (II)[(B1) M —L1] O —G—[(L2—(B2) K ] P

[0136] in

[0137] G comprises an oligonucleotide comprising at least two coding regions;

[0138] B1 is a position building unit, and M represents an integer from 1 to 20;

[0139] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and M is the same as or different from K;

[0140] L1 is a linker that operably connects B1 to G;

[0141] L2 is a linker that operably connects B2 to G;

[0142] O is 0 or 1;

[0143] P is 0 or 1;

[0144] The condition is that at least one of O and P is 1; and

[0145] Wherein each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G. In one embodiment, as described above, the benefits of the method of using the molecule of formula II may include the introduction of two coded parts: (B1) M and (B2) K OEM, which can increase the signal-to-noise ratio compared to OEM that displays a single encoded portion.

[0146] In one embodiment, the method may comprise one or more oligonucleotide encoding molecules having a structure according to formula (III),

[0147] (III)[(B1) M —L1] O —G'—[(L2—(B2) K ] P

[0148] in

[0149] G' comprises an oligonucleotide, G' comprising at least two coding regions and at least one hairpin region;

[0150] B1 is a position building unit, and M represents an integer from 1 to 20;

[0151] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0152] L1 is a linker that operably connects B1 to G';

[0153] L2 is a linker that operably connects B2 to G';

[0154] O is an integer from 0 to 5;

[0155] P is an integer from 0 to 5;

[0156] Provided that at least one of O and P is an integer from 1 to 5; and

[0157] Wherein each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G'. In one embodiment, as described above, the benefits of the method of using the molecule of formula II may include the introduction of 2 to 10 coded portions: (B1) M and (B2) K OEM, which can increase the signal-to-noise ratio compared to OEM that displays a single encoded portion.

[0158] In certain embodiments, the disclosure further relates to methods for forming oligonucleotide encoding molecules. In certain embodiments, the disclosure relates to methods for separating oligonucleotide encoding molecules by using affinity electrophoresis disclosed herein to determine the affinity of the encoded portion to the target molecule. In one embodiment, affinity electrophoresis can separate molecules based on desired properties, including but not limited to the ability to bind to the target molecule, the ability to bind to a specific region of the target molecule, the ability to competitively or non-competitively bind to known compounds, the ability not to bind to other anti-target molecules, the ability not to bind to other closely related target molecule categories or families, the ability to resist chemical changes produced by enzymes, the ability to resist chemical changes produced by enzyme families, the ability to be easily chemically altered by enzymes or enzyme families, the ability to have a certain degree of water solubility, the ability to have a certain degree of tissue permeability, and the ability to have a certain degree of cell permeability.

[0159] In certain embodiments, the molecule of formula (I) is an oligonucleotide encoding molecule. In one embodiment, the molecule of formula (II) and (III) is a subspecies of the molecule of formula (I). In one embodiment, the molecule of formula (III) is a subspecies of the molecule of formula (II). In certain embodiments of the molecule of formula (I), G comprises an oligonucleotide directed or selected for synthesizing the encoded portion. In certain embodiments of the molecule of formula (II) and (III), (B1) M and (B2) K Each represents the encoded portion. In some embodiments of formula (I) molecule, the molecule contains an oligonucleotide portion and at least one encoded portion. It should be understood that many structural features of the oligonucleotide in G are discussed herein with respect to the synthesis of at least one encoded portion of its guidance or encoding formula (I) molecule and the molecular structure relationship or the correlation of this synthesis process acting on the oligonucleotide encoding molecular structure. It should be understood that many structural features of the oligonucleotide in G or G' of the molecule of formula (I) or formula (II) and / or (III) are discussed according to the ability of the oligonucleotide in G or G', or its PCR copy, respectively, to identify, associate or promote the derivation of the synthesis step for preparing the molecule of formula (I). Therefore, it should be understood that there is a correlation between the sequence and / or structure of the building block of the encoded portion and the sequence or sequence combination of the coding region of the oligonucleotide portion. In one embodiment of the molecule of formula (I) and / or (II), G includes at least one hairpin region, and can be expressed as G'.

[0160] In certain embodiments of molecules of formula (I), (II) and (III), G or G' comprises or is an oligonucleotide. In certain embodiments, the oligonucleotide contains at least two coding regions, wherein 1% to 100%, including about 50% to 100%, including about 90% to 100% of the coding regions are single-stranded. In certain embodiments, the oligonucleotide in G or G' contains at least one terminal coding region, wherein one or both terminal coding regions are single-stranded. In certain embodiments, the oligonucleotide in G or G' contains at least one terminal coding region, wherein one or both terminal coding regions are double-stranded.

[0161] The term "hairpin structure" used in the present disclosure refers to a molecular structure containing 60% to 100% nucleotides by mass percentage and can hybridize with the terminal coding region of oligonucleotide G to form G'. In certain embodiments of the hairpin structure, the hairpin structure forms a single, continuous polymer chain and contains at least one overlapping portion (commonly referred to as a "stem"), wherein the overlapping portion contains a nucleotide sequence that hybridizes with the complementary sequence of the same hairpin structure. In certain embodiments of the hairpin structure, a bridge structure connects two separate oligonucleotide chains; the bridge structure may include a polyethylene glycol (PEG) polymer of 2 to 20 PEG units, including 3 to 15 PEG units, including 6 to 12 PEG units. In certain embodiments of the hairpin structure, the bridge structure may include an alkane chain of up to 30 carbons or a polyglycine chain of up to 20 units, or include some other chains with reactive functional groups.

[0162] In certain embodiments of molecules of formula (I), (II) and (III), the oligonucleotide in G or G' contains at least two coding regions, including 2 to about 21 coding regions, including 3 to 10 coding regions, including 3 to 5 coding regions. In certain embodiments, if the number of coding regions is less than 2, a combination of coding regions will be possible. In certain embodiments, if the number of coding regions exceeds 20, inefficient synthesis will interfere with accurate synthesis.

[0163] In certain embodiments of molecules of formula (I), (II) and (III), about 50% to 100% of at least two coding regions contain about 6 to about 50 nucleotides, including about 12 to about 40 nucleotides, including about 8 to about 30 nucleotides. In certain embodiments, if the coding region comprises less than about 6 nucleotides, the coding region cannot accurately guide the synthesis of the encoded portion. In certain embodiments, if the coding region contains more than about 50 nucleotides, the coding region will be cross-reactive. This cross-reactivity can interfere with the ability of the coding region to accurately guide and identify the synthesis steps for synthesizing the encoded portion of the molecules of formula (I), (II) and (III).

[0164] In certain embodiments of molecules of formula (I), (II) and (III), the purpose of the oligonucleotide in G or G' is to direct the synthesis of at least one encoded portion of a molecule of formula (I), (II) or (III) by selective hybridization with a complementary anti-coding strand. In certain embodiments, the coding region is single-stranded to facilitate hybridization with the complementary strand. In certain embodiments, 70% to 100%, including 80% to 99%, including 80% to 95%, of the coding region is single-stranded. It should be understood that the complementary strand of the coding region, if present, can be added after the step of encoding the encoded portion of the molecule of formula (I), (II) and (III) during synthesis.

[0165] In certain embodiments, oligonucleotides may contain natural and non-natural nucleotides. Suitable nucleotides include natural nucleotides of DNA (deoxyribonucleic acid), including adenine (A), guanine (G), cytosine (C) and thymine (T), and natural nucleotides of RNA (ribonucleic acid), adenine (A), uracil (U), guanine (G) and cytosine (C). Other suitable bases include natural bases such as deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine, inosine, diaminopurine; base analogs such as 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 4-((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)amino)pyrimidine-2(1H )-one, 4-amino-5-(hept-1,5-diyn-1-yl)pyrimidin-2(1H)-one, 6-methyl-3,7-dihydro-2H-pyrrolo[2,3-d]pyrimidin-2-one, 3H-benzo[b]pyrimidin[4,5-e][1,4]oxazin-2(10H)-one and 2-thiocytidine; modified nucleotides, such as 2'-substituted nucleotides, including 2'-O-methylated bases and 2'-fluoro bases; and modified sugars, such as 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, hexose; and / or modified phosphate groups, such as thiophosphate and 5'-N-phosphoramidite linkages. It should be understood that an oligonucleotide is a polymer of nucleotides. The terms "polymer" and "oligomer" are used interchangeably herein. In certain embodiments, an oligonucleotide does not necessarily contain consecutive bases. In certain embodiments, oligonucleotides may be interspersed with linker moieties or non-nucleotide molecules.

[0166] In certain embodiments of the molecules of formula (I), (II), and (III), the oligonucleotide in G contains about 60% to 100%, including about 80% to 99%, including about 80% to 95% DNA nucleotides. In certain embodiments, the oligonucleotide contains about 60% to 100%, including about 80% to 99%, including about 80% to 95% RNA nucleotides.

[0167] In certain embodiments of molecules of formula (I), (II) and (III), the oligonucleotide in G or G' contains at least two coding regions, wherein at least two coding regions overlap so as to be coextensive, provided that the overlapping coding regions share only about 30% to 1%, including about 20% to 1%, including about 10% to 2%, of the same nucleotides. In certain embodiments of molecules of formula (I), (II) and (III), the oligonucleotide in G or G' is about 40% to 100%, including about 60% to 100%, including about 80% to 100% single stranded. In certain embodiments of molecules of formula (I), (II) and (III), the oligonucleotide in G or G' contains at least two coding regions, wherein at least two coding regions are adjacent. In certain embodiments of molecules of formula (I), (II) and (III), the oligonucleotide in G or G' contains at least two coding regions, wherein at least two coding regions are separated by a region of nucleotides that does not direct or record the synthesis of the encoded portion of the molecule of formula (I), (II) or (III).

[0168] The term "non-coding region", when present, refers to a region of an oligonucleotide that is incapable of hybridizing to a complementary strand of nucleotides to direct the synthesis of the encoded portion of a molecule of Formula (I), (II) and (III), or does not correspond to any anti-coding oligonucleotide used to sort the molecules of Formula (I), (II) and (III) during synthesis. In certain embodiments, the non-coding region is optional. In certain embodiments, the oligonucleotide contains 1 to about 20 non-coding regions, including 2 to about 9 non-coding regions, including 2 to about 4 non-coding regions. In certain embodiments, the non-coding region contains about 4 to about 50 nucleotides, including about 12 to about 40 nucleotides, and including about 8 to about 30 nucleotides.

[0169] In certain embodiments of molecules of formula (I), (II) and (III), one purpose of the non-coding region is to separate the coding region to avoid or reduce cross-hybridization, because cross-hybridization can interfere with the accurate coding of the encoded portion of the molecules of formula (I), (II) and (III). In certain embodiments, one purpose of the non-coding region is to add functionality to the molecules of formula (I), (II) and (III), rather than just hybridization or coding. In certain embodiments, one or more non-coding regions can be regions of oligonucleotides modified with markers, such as fluorescent markers or radioactive markers. Such markers can promote visualization or quantification of molecules of formula (I), (II) and (III). In certain embodiments, one or more non-coding regions are modified with functional groups or tethers that are conducive to processing. In certain embodiments, one or more non-coding regions are double-stranded, which reduces cross-hybridization. In certain embodiments, it should be understood that the non-coding region is optional. In certain embodiments, suitable non-coding regions do not interfere with PCR amplification of oligonucleotides.

[0170] In some embodiments, one or more coding regions may be regions of an oligonucleotide in G or G' modified with a label, such as a fluorescent label or a radioactive label. Such labels may facilitate visualization or quantification of molecules of formula (I), (II) and (III). In some embodiments, one or more coding regions are modified with a functional group or tether that facilitates processing.

[0171] In certain embodiments of the molecules of formula (I), (II) and (III), G or G' comprises a compound of formula (C N —(Z N —C N+1 ) A ) or (Z N —(C N —Z N+1 ) A ), wherein C is a coding region, Z is a non-coding region, N is an integer from 1 to 20, and A is an integer from 1 to 20; wherein each non-coding region contains 0 to 50 nucleotides and is optionally double-stranded. In certain embodiments of molecules of formula (I), (II) and (III), each or most of the coding regions contain 6 to 50 nucleotides. In certain embodiments of molecules of formula (I), (II) and (III), each or most of the coding regions contain 8 to 30 nucleotides.

[0172] In certain embodiments of molecules of Formula (I), (II) and (III), about 10% to 100% of the positional building block B1 at position M and / or B2 at position K are associated with a combination of 2, 3, 4 or 5 coding regions, including about 20% to 100%, including about 30% to 100%, including about 50% to 100%, including about 70% to 100%, including about 90% to 100%. In contrast, in certain embodiments of molecules of Formula (I), (II) and (III), 0 to about 90% of the positional building block B1 at position M and / or B2 at position K are associated with or identified by a single coding region, including 0 to about 10%, including 0 to about 20%, including 0 to about 30%, including 0 to about 50%, including 0 to about 70%.

[0173] In certain embodiments of the molecules of formula (I), (II) and (III), B represents a positional building block. As used herein, the phrase "building block" or "positional building block" refers to a unit in a series of individual building blocks that are joined together as subunits to form a larger molecular structure. In certain embodiments, (B1) M and (B2) K Each independently represents a series of individual building blocks that are bonded together to form a polymer chain having a number of units M and K, respectively. For example, where M is 10, then (B) 10 Refers to a series of building blocks: B 10 —B9—B8—B7—B6—B5—B4—B3—B2—B1. For example, where M is 3 and K is 2, formula (I) can be accurately expressed as the following formula:

[0174] [((B1)3—(B1)2—(B1)1—L1] O —G—[(L2—(B2)1—(B2)2] P .

[0175] It is to be understood that M and K are each independently used as positional identifiers for each individual unit of B, and that the "1" or "2" of B1 or B2 is used merely to distinguish which chain is being referred to.

[0176] The precise definition of the term "building block" in this disclosure depends on its context. "Building block" is a chemical structural unit that can be chemically connected to other chemical structural units. In some embodiments, the building block has one, two or more reactive chemical groups that allow the building block to undergo a chemical reaction that connects the building block to other chemical structural units. It should be understood that when the building block reacts to form a chemical bond, some or all of the reactive chemical groups of the building block may be lost. For example, the building block in the solution may have two reactive chemical groups. In this example, the building block in the solution can react with the reactive chemical groups of the building block that is part of the chain of the building block to increase the length of the chain or extend a branched chain from the chain. When the building block is mentioned in the case of a solution or as a reactant, the building block will be understood to include at least one reactive chemical group but may include two or more reactive chemical groups. When the building block is mentioned in the case of a polymer, oligomer or molecule larger than the building block itself, the building block will be understood to have a structure of the building block as a (monomer) unit of a larger molecule, even if one or more chemical reactive groups will have reacted.

[0177] The type of molecule or compound that can be used as building block is generally not limited, as long as a kind of building block can react with another building block to form a covalent bond. In some embodiments, building block has a chemically reactive group used as terminal unit. In some embodiments, building block has 1,2,3,4,5 or 6 suitable reactive chemical groups. In some embodiments, the position building block of B has 1,2,3,4,5 or 6 suitable reactive chemical groups independently of each other. Suitable reactive chemical groups for building block include primary amine, secondary amine, carboxylic acid, thioacid, primary alcohol, secondary alcohol, ester, thiol, isocyanate, isothiocyanate, chloroformate, sulfonyl chloride, sulfonyl fluoride, thiocarbonate, heteroaryl halide, aldehyde, ketone, haloacetate, aryl halide, azide, halide, triflate, diene, dienophile, boric acid, boric ester, α-β unsaturated ketone, cyanoacrylamide, maleimide, alkyne and alkene.

[0178] Any coupling chemistry can be used to link the building blocks, provided that the coupling chemistry is compatible with the presence of the oligonucleotide. Exemplary coupling chemistries include the formation of amides by reaction of amines, such as DNA-linked amines, with Fmoc-protected amino acids or other various substituted carboxylic acids; the formation of ureas by reaction of amines (including DNA-linked amines) with isocyanates and another amine (ureation); the formation of carbamates by reaction of amines (including DNA-linked amines) with chloroformates (carbamoylation) and alcohols; the formation of sulfonamides by reaction of amines (including DNA-linked amines) with sulfonyl chlorides; the formation of thioureas by reaction of amines (including DNA-linked amines) with thiocarbonates and another amine (thioureation); the formation of anilines by reaction of amines (including DNA-linked amines) with heteroaryl halides (SNAr); the formation of secondary amines by reaction of amines (including DNA-linked amines) with aldehydes followed by reduction (reductive amination); the formation of peptoids by acylation of amines (including DNA-linked amines) with chloroacetates followed by displacement of the chloride with another amine (SN2 reaction); the formation of acyl groups by acylating amines (including DNA-linked amines) with carboxylic acids substituted with aryl halides. Alkyne-containing compounds are formed by acylation of amines (including DNA-linked amines) followed by substitution of the halide with a substituted alkyne (Sonogashira reaction); biaryl compounds are formed by acylation of amines (including DNA-linked amines) with carboxylic acids substituted with aryl halides followed by substitution of the halide with a substituted boronic acid (Suzuki reaction); substituted triazines are formed by reaction of amines (including DNA-linked amines) with cyanuric chloride followed by reaction with another amine, phenol or thiol (cyanurylation, aromatic substitution); secondary amines are formed by acylation of amines (including DNA-linked amines) with carboxylic acids substituted with an appropriate leaving group such as a halide or triflate followed by substitution of the leaving group with another amine (SN2 / SN1 reaction); and cyclic compounds are formed by substitution of amines with compounds bearing olefins or alkynes and reaction of the product with an azide or olefin (Diels-Alder and Huisgen reactions). In certain embodiments of the reaction, the molecules that react with the amine group, including primary amines, secondary amines, carboxylic acids, primary alcohols, esters, thiols, isocyanates, chloroformates, sulfonyl chlorides, thiocarbonates, heteroaryl halides, aldehydes, chloroacetates, aryl halides, olefins, halides, boronic acids, alkynes, and alkenes have a molecular weight of about 30 to about 500 Daltons.

[0179] In certain embodiments of the coupling reaction, any chemistry of molecules with secondary reactive groups such as amines, thiols, halides, boronic acids, alkynes, or alkenes can be used to add the first building block by replacing the amine, including the amine to which DNA is attached. The secondary reactive group can then react with a building block with an appropriate reactive group. Exemplary secondary reactive group coupling chemistries include acylation of amines (including DNA-linked amines) with Fmoc-amino acids, followed by removal of the protecting group and reductive amination of the newly deprotected amine with an aldehyde and borohydride; reductive amination of amines (including DNA-linked amines) with aldehydes or ketones and borohydride, followed by reaction of the now substituted amine with cyanuric chloride, followed by displacement of the other chloride from the triazine with a thiol, phenol, or another amine; acylation of amines (including DNA-linked amines) with carboxylic acids substituted with heteroaryl halides, followed by SNAr reaction with another amine or thiol to displace the halide and form an aniline or thioether; acylation of amines (including DNA-linked amines) with carboxylic acids substituted with halogenated aromatic groups, followed by displacement of the halide by an alkyne in a Sonogashira reaction; or displacement of the halide by an aryl in a boronate-mediated Suzuki reaction.

[0180] In certain embodiments, the coupling chemistry is based on suitable bonding reactions known in the art. See, e.g., March, Advanced Organic Chemistry, 4th Edition, New York: John Wiley and Sons (1992), Chapters 10 to 16; Carey and Sundberg, Advanced Organic Chemistry, Part B, Plenum (1990), Chapters 1 to 11; and Coltman et al., Principles and Applications of Organotransition MetalChemistry, University Science Books, Mill Valley, Calif. (1987), Chapters 13 to 20; the entire contents of which are incorporated herein by reference.

[0181] In some embodiments, in addition to one or more reactive groups for connecting building blocks, building blocks can also include one or more functional groups. One or more of these other functional groups can be protected to prevent these functional groups from undesirable reactions. Suitable protecting groups for various functional groups are known in the art (Greene and Wuts, Protective Groups in Organic Synthesis, Second Edition, New York: John Wiley and Sons (1991), the entire contents of which are incorporated herein by reference). Particularly useful protecting groups include tert-butyl esters and ethers, acetals, trityl ethers and amines, acetyl esters, trimethylsilyl ethers, trichloroethyl ethers and esters and carbamates.

[0182] The type of building block is generally not limited, as long as the building block is compatible with one or more reactive groups that can form covalent bonds with other building blocks. Suitable building blocks include, but are not limited to, peptides, carbohydrates, glycolipids, lipids, proteoglycans, glycopeptides, sulfonamides, nucleoproteins, ureas, carbamates, vinyl polypeptides, amides, vinyl sulfonamide peptides, esters, carbohydrates, carbonates, peptidyl phosphonates, azatides, peptoids (oligomeric N-substituted glycines), ethers, ethoxyformacetal oligomers, thioethers, ethylene, ethylene glycol, disulfides, arylene sulfides, nucleotides, morpholinos, imines, pyrrolidones, ethyleneimines, acetates, styrenes, acetylene, vinyl, phospholipids, siloxanes, isocyanides, isocyanates, and methacrylates. In certain embodiments, (B1) of formula (I) M or (B2) K Each independently represents a polymer having these building blocks of M or K units, respectively, including polypeptides, polysaccharides, polyglycolipids, polylipids, polyproteoglycans, polyglycopeptides, polysulfonamides, polynuclear proteins, polyureas, polyurethanes, polyvinyl analog polypeptides, polyamides, polyvinyl analog sulfonamide peptides, polyesters, polysaccharides, polycarbonates, polypeptidylphosphonates, polyazatides, polypeptide-like proteins (oligomeric N-substituted glycines), polyethers, polythoxyformacetal oligomers, polythioethers, polyethylene, polyethylene glycol, polydisulfides, polyarylether sulfides, polynucleotides, polymorpholinos, polyimines, polypyrrolinones, polyethyleneimines, polyacetates, polystyrenes, polyacetylenes, polyethylenes, polyphospholipids, polysiloxanes, polyisocyanides, polyisocyanates, and polymethacrylates. In certain embodiments of molecules of Formula (I), about 50% to about 100%, including about 60% to about 95%, and including about 70% to about 90% of the building blocks have a molecular weight of about 30 to about 500 Daltons, including about 40 to about 350 Daltons, including about 50 to about 200 Daltons.

[0183] It should be understood that a building block with two reactive groups will form a linear oligomeric or polymeric structure, or a linear non-polymeric molecule, comprising each building block as a unit. It should also be understood that a building block with three or more reactive groups can form a molecule with branches at each building block with three or more reactive groups.

[0184] In certain embodiments of the molecules of formula (I), (II) or (III), L, L1 and L2 each independently represent a joint. The term "joint molecule" refers to a molecule having two or more reactive groups that can react to form a joint. The term "joint" refers to a portion of a molecule that operably connects or covalently bonds a hairpin structure of G or G' to a building block. The term "operably connected" refers to two or more chemical structures that are connected or covalently bound together in a manner that remains connected throughout the various operations (including PCR amplification) that the oligonucleotide encoding molecule is expected to experience.

[0185] In certain embodiments of the molecules of formula (II) or (III), L l is to respectively operate B lLinkers connected to G or G'. In certain embodiments of the molecules of formula (II) or (III), L2 is a linker that operably connects B2 to G or G', respectively. In certain embodiments, L1 and L2 are each independently a bifunctional molecule that connects B1 to G or G' by reacting one of the reactive functional groups of L1 with a reactive group of B1 and another reactive functional group of L1 with a reactive functional group of G or a hairpin region of G', or reacting one of the reactive functional groups of L2 with a reactive group of B2 and another reactive functional group of L2 with a reactive functional group of G or a hairpin region of G', in no particular order. In certain embodiments of the molecule of the hairpin region of G', L1 and L2 are each independently formed by chemically reactive groups of B1 and G or B2 and G and a PEG (e.g., azido-PEG-NHS, or azido-PEG-amine, or diazido-PEG), or an alkane acid chain portion (e.g., 5-azidopentanoic acid, (S)-2-(azidomethyl)-1-Boc-pyrrolidine, 4-azidoaniline or 4-azido-butane-1-acid N-hydroxysuccinimide ester); a thiol-reactive linker, such as those that are PEG (e.g., SM(PEG)n NHS-PEG-maleimide), alkane chains (e.g., 3-(pyridin-2-yldisulfanyl)-propionic acid-Osu or sulfosuccinimidyl 6-(3'-[2-pyridyldisulfanyl]-propionamido)hexanoate); and amides for oligonucleotide synthesis, such as amino modifiers (e.g., 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite), thiol modifiers (e.g., 5-trityl-6-mercaptohexyl- 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or a chemically co-reactive modifier (e.g., 6-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 3-dimethoxytrityloxy-2-(3-(3-propargyloxypropionamido)propionamido)propyl-1-O-succinyl, long chain alkylamino CPG or 4-azido-butane-1-acid N-hydroxysuccinimide ester); and compatible combinations thereof.

[0186] In certain embodiments of molecules of formula (III), the hairpin region of G' may be annotated as H1 or H2, wherein each hairpin region independently comprises from about 20 to about 90 nucleotides, including from about 32 to about 80 nucleotides, including from about 45 to about 80 nucleotides. In certain embodiments, H1 and H2 each independently contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including from 1 to 5, including from 2 to 4, including from 2 to 3 nucleotides modified with appropriate functional groups for promoting reaction with linker molecules or optionally with building blocks, including where H1 and H2 are each independently synthesized using a base, such as, but not limited to, 5'-dimethoxytrityl-5-ethynyl-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as 5-ethynyl-dU-CE phosphoramidite, available from Glen Research, Sterling VA). In certain embodiments, H1 and H2 each independently comprise a non-nucleotide having a suitable functional group that facilitates reaction with a linker molecule or optionally with a building block, including but not limited to 3-dimethoxytrityloxy-2-(3-(5-hexynylamido)propynylamido)propyl-1-O-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as alkyne-modified serinol phosphoramidite, from Glen Research, Sterling VA) and abasic alkyne CEP (from IBA GmbH, Gottingen, Germany). In certain embodiments, H l H and H2 each independently include nucleotides having a modified base that already has a linker, such as H land H2 can each be synthesized using a base such as, but not limited to, 5'-dimethoxytrityl-N6-benzoyl-N8-[6-(trifluoroacetylamino)-hex-1-yl]-8-amino-2'-deoxyadenosine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as amino modifier C6 dA, available from Glen Research, Sterling VA), 5'-dimethoxytrityl-N2-[6-(trifluoroacetylamino)-hex-1-yl]-2'-deoxyguanosine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as amino modifier C6 dG, available from Glen Research, Sterling VA). Research, Sterling, VA), 5'-dimethoxytrityl-5-[3-methyl-acryl]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as CarboxydT, purchased from Glen Research, Sterling, VA), 5'-dimethoxytrityl-5-[N-((9-fluorenylmethoxycarbonyl)-aminohexyl)-3-acrylimide]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as Fmoc-amino modifier C6 dT, Glen Research, Sterling, VA), 5'-dimethoxytrityl-5-(oct-1,7-diynyl)-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as C8 alkyne dT, Glen Research, Sterling VA), 5'-(4,4'-dimethoxytrityl)-5-[N-(6-(3-benzoylthiopropionyl)-aminohexyl)-3-acrylamide]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (also known as S-Bz-thiol-modifier C6-dT, Glen Research, Sterling VA), and 5-carboxy dC CEP (from IBA GmbH, Göttingen, Germany), N4-TriGl-amino 2'deoxycytidine (from IBA GmbH, Göttingen, Germany). Suitable functional groups for modifying nucleotides and non-nucleotides in H1 and H2 include, but are not limited to, primary amines, secondary amines, carboxylic acids, primary alcohols, esters, thiols, isocyanates, chloroformates, sulfonyl chlorides, thiocarbonates, heteroaryl halides, aldehydes, chloroacetates, aryl halides, halides, boronic acids, alkynes, azides, and olefins.

[0187] In certain embodiments, one or more hairpin structures H1 and H2 can be modified with a label, such as a fluorescent label or a radioactive label. Such a label can facilitate visualization or quantification of the molecule of formula (III). In certain embodiments, one or more hairpin structures H1 and H2 are modified with a functional group or tether that facilitates processing.

[0188] In certain embodiments of the molecule of formula (III), H l The benefit of the hairpin structure of H2 and H3 is that one or both can allow the multi-display (polydisplay) of multiple coding parts at one or both ends of the molecule of formula (III). Without wishing to be bound by theory, it is believed that the multi-display of multiple encoded parts at one or both ends of the oligonucleotide encoding molecule disclosed herein provides improved selection characteristics under certain conditions. For example, the multivalent display of the encoded compound can increase the apparent affinity through the affinity effect.

[0189] In certain embodiments of molecules of formula (II) or (III), about 10% to 100% of the positional building blocks B1 at position M and / or B2 at position K are associated with a combination of 2, 3, 4 or 5 coding regions, including about 20% to 100%, including about 30% to 100%, including about 50% to 100%, including about 70% to 100%, including about 90% to 100%. In contrast, in certain embodiments of molecules of formula (II) or (III), 0 to about 90% of the positional building blocks B1 at position M and / or B2 at position K are associated with or identified by a single coding region, including 0 to about 10%, including 0 to about 20%, including 0 to about 30%, including 0 to about 50%, including 0 to about 70%.

[0190] The present disclosure relates to a method for synthesizing oligonucleotide encoding molecules, including molecules of formula (I), (II) and (III). In certain embodiments of the method for synthesizing molecules of formula (I), (II) and (III), the method uses a series of "sorting (sort and reaction) " steps, wherein a mixture of oligonucleotide encoding molecules containing different coding region combinations is sorted into a sub-pool by selective hybridization of one or more coding regions of the oligonucleotide encoding molecules with an anti-coding oligomer fixed on a hybridization array. In certain embodiments of the method, the benefit of sorting the oligonucleotide encoding molecules into sub-pools is that this separation allows each sub-pool to react with a positional building block B including B1 and / or B2 under separate reaction conditions before combining or mixing the sub-pools of the oligonucleotide encoding molecules for further chemical treatment. In certain embodiments of the method, the sorting and reaction process can be repeated to add a series of positional building blocks. In certain embodiments of the method, a benefit of using a sort and react method to add building blocks is that the identity of the building block at each position of the encoded portion of the molecule can be associated with 1, 2, 3, 4 or 5 coding regions used to selectively isolate or sort the oligonucleotide encoding molecules prior to adding the building blocks.

[0191] In certain embodiments, one or more building blocks can be added by using a single sorting step to separate the oligonucleotide encoding molecules into subpools, reacting the oligonucleotide encoding molecules with the building blocks, and then remixing. In such embodiments, one coding region used to sort the oligonucleotide encoding molecules during synthesis will uniquely identify or associate to the building block based on its position, because the characteristics of the coding region used can be associated with the characteristics of the reaction used to add the building block, which will include the characteristics of the position of the added building block.

[0192] In certain embodiments, one or more building blocks can be added by 2, 3, 4, or 5 sorting steps, reacting the oligonucleotide encoding molecules with the building blocks, and then mixing. In such embodiments, the combination or series of coding regions used to sort the oligonucleotide encoding molecules during synthesis will uniquely identify or be associated with the building blocks based on their position, because the combination or series of coding regions used can be associated with the characteristics of the reaction used to add the building blocks, which will include the characteristics or structure of the location of the added building blocks.

[0193] In certain embodiments, the synthesis method can be independently converted from a single sorting step (single expression) or a series of sorting steps (multiple expressions) as needed. In certain embodiments of the method, about 10% to 100% of the positional building block B1 at position M and / or B2 at position K are added through a series of 2, 3, 4 or 5 sorting steps, including about 20% to 100%, including about 30% to 100%, including about 50% to 100%, including about 70% to 100%, including about 90% to 100%. If the amount of positional building blocks added using a series of sorting steps is less than 10%, the benefits of low cost and more efficient synthesis will not be appreciated.

[0194] It should be understood that the molecules of formula (I), (II) and (III) may include one or more coding regions that are identical between or among the molecules in the library (pool), but it should also be understood that the vast majority, if not all, of the molecules in the library will have different coding region combinations. In some embodiments of the method, the benefit of a molecule library with different coding region combinations is that different combinations can encode oligonucleotide encoding molecules with a plurality of different encoded portions.

[0195] In certain embodiments, the synthesis method includes providing at least one hybridization array. The step of providing the hybridization array is generally not limited, and includes using techniques known in the art or commercially purchasing the hybridization array to manufacture the hybridization array. In certain embodiments of the method, the hybridization array includes a substrate of at least two separate regions, having fixed anticodon oligomers on its surface. In certain embodiments, each region of the hybridization array contains different immobilized anticodon oligomers, wherein the anticodon oligomer is an oligonucleotide sequence capable of hybridizing with one or more coding regions of the molecules of formula (I) including formula (II) and (III). In certain embodiments of the method, the hybridization array uses two or more chambers. In certain embodiments of the method, the chamber of the hybridization array contains particles, such as beads, which have fixed anticodon oligomers on the particle surface. In certain embodiments of the method, the benefit of fixing the molecules of formula (I) including formula (II) and (III) on the array is that the step allows the molecules to be sorted or selectively separated into sub-libraries of molecules based on the specific oligonucleotide sequences of one or more coding regions. In certain embodiments, the separated molecular sub-libraries can then be released or removed from the array individually into the reaction chamber for further hybridization steps or chemical reaction treatments. In certain embodiments, the release step is optional, generally without limitation, and may include dehybridizing the molecules by heating, using a denaturing agent, or exposing the molecules to a buffer having a pH ≥ 12. In certain embodiments, chambers or regions of the array containing different immobilized oligonucleotides may be positioned to allow the contents of each chamber or region to flow into an array of wells for further chemical processing.

[0196] In some embodiments, the method includes reacting at least one building block B, including B1 and / or B2, with an oligonucleotide encoding molecule to form a sub-library of molecules of formula (II) and (III), wherein B1 and / or B2 are as defined above for formula (II) and (III). In some embodiments, building blocks B1 and / or B2 can be added to a container before, during or after the molecules of formula (II) and (III). It should be understood that the container can contain solvents and co-reactants under acidic, alkaline or neutral conditions, depending on the chemistry used to react with the oligonucleotide encoding molecule and covalently link building blocks B1 and / or B2 to form molecules of formula (II) and (III).

[0197] In some embodiments of the method, the amplification step includes using PCR techniques known in the art to produce a copy sequence of the oligonucleotide in G or G' of formula (I), (II) and (III) respectively. In some embodiments of the method, the copy sequence contains a copy of at least two coding regions of formula (I), (II) and (III). In some embodiments, a benefit of amplifying the oligonucleotide in G or G' from at least one probe molecule includes being able to detect which coding portions of the oligonucleotide encoding molecule can bind to the target molecule, even if the oligonucleotide encoding molecule cannot be easily removed from the target molecule. In some embodiments, the benefit of amplification is that it allows the generation of a molecular library with huge diversity. This huge diversity is at the expense of any given formula (I), (II) and (III) molecule in low numbers. PCR amplification allows the identification of oligonucleotide sequences present in very small quantities by increasing the number until a quantity that is easy to detect is reached. Then, DNA sequencing and analysis of the copy sequence can identify or associate the encoded portions of the oligonucleotide encoding molecules of formula (I), (II) and (III) that can bind to the target.

[0198] The synthesis and analysis of molecular libraries of formula (I), (II) and (III) or identifiable variants thereof have been previously disclosed in WO2017 / 218293, WO 2018 / 204420 and PCT / US2018 / 052494 (not published at the time of filing), the entireties of which are incorporated herein by reference.

[0199] Exemplary Embodiments

[0200] Embodiment 1. A method for collecting target activity data of at least one resolved oligonucleotide-encoded molecule, comprising:

[0201] providing a separation medium, wherein the separation medium contains at least one target molecule;

[0202] introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules into a separation medium, wherein the at least two different oligonucleotide encoding molecules include an encoding portion operably linked to at least one encoded portion;

[0203] forming at least two different resolved oligonucleotide encoding molecules by separating the at least two different oligonucleotide encoding molecules into at least two separate locations in a separation medium;

[0204] harvesting at least one resolved oligonucleotide encoding molecule from at least two different resolved oligonucleotide encoding molecules by fragmenting at least one of the at least two separate locations from a separation medium to form at least one resolved fragment;

[0205] processing at least one resolved oligonucleotide encoding molecule to allow for PCR;

[0206] amplifying at least one encoded portion of the at least one resolved oligonucleotide encoding molecule by performing PCR on the encoding portion of the at least one resolved oligonucleotide encoding molecule; and

[0207] Target activity data for at least one resolved oligonucleotide encoding molecule is collected by correlating at least one position with a feature of at least one encoded portion of the at least one resolved oligonucleotide encoding molecule.

[0208] Embodiment 2. The method according to any one of embodiments 1 or 3-15, wherein the at least one target molecule comprises at least one of a cell, an oligonucleotide, a protein, an enzyme, a ribosome, and a nanodisc.

[0209] Embodiment 3. A method according to any one of embodiments 1-2 or 4-15, wherein the separation medium contains at least one of particles, polymers and separation surfaces, and the at least one target molecule is connected to at least one of the separation medium, particles, polymers and separation surfaces.

[0210] Embodiment 4. The method of any one of embodiments 1-3 or 5-15, wherein the particles comprise polymer particles or metal colloids.

[0211] Embodiment 5. The method of any one of embodiments 1-4 or 6-15, wherein the polymer has a molecular weight of 10% or more of the lowest weight target molecule of the at least one target molecule.

[0212] Embodiment 6. The method according to any one of embodiments 1-5 or 7-15, wherein at least two different oligonucleotide encoding molecules are separated based on at least one target activity between at least one target molecule and the encoded portions of the at least two different oligonucleotide encoding molecules.

[0213] Embodiment 7. A method according to any one of embodiments 1-6 or 8-15, wherein the at least one target activity comprises chemically modifying the encoded portion of the at least one oligonucleotide encoding molecule by the at least one target molecule.

[0214] Embodiment 8. The method according to any one of embodiments 1-7 or 9-15, wherein

[0215] The oligonucleotide contains at least two coding regions,

[0216] at least one encoding portion comprises at least two position building blocks,

[0217] Each position building block of at least one encoded portion is identified by 1 to 5 coding regions of the oligonucleotide; and

[0218] The separation medium contains a porous gel and a buffer system.

[0219] Embodiment 9. The method according to any one of embodiments 1-8 or 10-15, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (I),

[0220] (I)G-L-B

[0221] in

[0222] G comprises an oligonucleotide comprising at least two coding regions;

[0223] B is an encoded portion containing at least two building blocks;

[0224] L is a linker that operably connects G to B; and

[0225] Each position building block in B is identified according to the position of 1 to 5 coding regions of G.

[0226] Embodiment 10. The method according to any one of embodiments 1-9 or 11-15, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (II),

[0227] (II)[(B1) M —L1] O —G—[(L2—(B2) K ] P

[0228] in

[0229] G comprises an oligonucleotide comprising at least two coding regions;

[0230] B1 is a position building unit, and M represents an integer from 1 to 20;

[0231] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0232] L1 is a linker that operably connects B1 to G;

[0233] L2 is a linker that operably connects B2 to G;

[0234] O is zero or 1;

[0235] P is zero or 1;

[0236] The condition is that at least one of O and P is 1; and

[0237] Wherein each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G.

[0238] Embodiment 11. The method according to any one of embodiments 1-10 or 12-15, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (III),

[0239] (III)[(B1) M —L1] O —G'—[(L2—(B2) K ] P

[0240] in

[0241] G' comprises an oligonucleotide, G' comprising at least two coding regions and at least one hairpin region;

[0242] B1 is a position building unit, and M represents an integer from 1 to 20;

[0243] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0244] L1 is a linker that operably connects B1 to G';

[0245] L2 is a linker that operably connects B2 to G';

[0246] O is an integer from 0 to 5;

[0247] P is an integer from 0 to 5;

[0248] Provided that at least one of O and P is an integer from 1 to 5; and

[0249] Each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G'.

[0250] Embodiment 12. The method according to any one of embodiments 1-11 or 13-15, further comprising:

[0251] separating at least two different oligonucleotide encoding molecules into at least two separate locations in the separation medium by applying a first separation treatment in a first direction across the separation medium,

[0252] The first separation process includes a first voltage scheme and a first duration.

[0253] Embodiment 13. The method according to any one of embodiments 1-12 or 14-15, further comprising:

[0254] At least one resolved oligonucleotide-encoded molecule is harvested by fragmenting at least one location from the separation medium in a first fragmentation direction substantially perpendicular to the first direction to form at least one resolved fragment.

[0255] Embodiment 14. The method according to any one of embodiments 1-13 or 15, further comprising:

[0256] separating the at least two different oligonucleotide encoding molecules into at least two separate locations of the separation medium by applying a second separation treatment across the separation medium in a second direction, wherein the second direction is substantially perpendicular to the first direction,

[0257] The second separation process includes a second voltage scheme and a second duration.

[0258] Embodiment 15. The method according to any one of embodiments 1-14, further comprising:

[0259] At least one resolved oligonucleotide-encoded molecule is harvested by fragmenting at least one location from the separation medium in a second fragmentation direction substantially perpendicular to the first fragmentation direction to form at least one resolved fragment.

[0260] Even More Exemplary Embodiments

[0261] Embodiment 1A. A method comprising:

[0262] providing a separation medium, wherein the separation medium contains at least one target molecule and at least one sample region;

[0263] introducing a sample containing a mixture of at least two different oligonucleotide encoding molecules comprising an encoding portion operably linked to an encoded portion into a sample zone of the separation medium; and

[0264] At least a portion of the mixture is contacted with the target in a separation medium by subjecting the sample to electrophoresis.

[0265] Embodiment 2A. The method of embodiment 1A, wherein the encoding portion comprises an oligonucleotide linked to at least one encoded portion,

[0266] wherein the oligonucleotide contains at least two coding regions,

[0267] wherein at least one of the encoded portions contains at least two position building blocks,

[0268] wherein each position building block of at least one of the encoded portions is identified by 1 to 5 coding regions; and

[0269] The separation medium contains a porous gel and a buffer system.

[0270] Embodiment 3A. The method of embodiment 1A, wherein the at least one target molecule comprises at least one of a cell, an oligonucleotide, a protein, an enzyme, a ribosome, and a nanodisc.

[0271] Embodiment 4A. The method of embodiment 1A, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (I),

[0272] (I)G-L-B

[0273] in

[0274] G comprises an oligonucleotide comprising at least two coding regions;

[0275] B is an encoded portion containing at least two building blocks;

[0276] L is a linker that operably connects G to B; and

[0277] Each position building block in B is identified according to the position of 1 to 5 coding regions of G.

[0278] Embodiment 5A. The method of embodiment 1A, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (II),

[0279] (II)[(B1) M —L1] O —G—[(L2—(B2) K ] P

[0280] in

[0281] G comprises an oligonucleotide comprising at least two coding regions;

[0282] B1 is a position building unit, and M represents an integer from 1 to 20;

[0283] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0284] L1 is a linker that operably connects B1 to G;

[0285] L2 is a linker that operably connects B2 to G;

[0286] O is zero or 1;

[0287] P is zero or 1;

[0288] The condition is that at least one of O and P is 1; and

[0289] Wherein each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G.

[0290] Embodiment 6A. The method of embodiment 1A, wherein the at least two different oligonucleotide encoding molecules have a structure according to formula (III),

[0291] (III)[(B1) M —L1] O —G'—[(L2—(B2) K ] P

[0292] in

[0293] G' comprises an oligonucleotide, G' comprising at least two coding regions and at least one hairpin region;

[0294] B1 is a position building unit, and M represents an integer from 1 to 20;

[0295] B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K;

[0296] L1 is a linker that operably connects B1 to G';

[0297] L2 is a linker that operably connects B2 to G';

[0298] O is an integer from 0 to 5;

[0299] P is an integer from 0 to 5;

[0300] Provided that at least one of O and P is an integer from 1 to 5; and

[0301] Each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G'.

[0302] Embodiment 7A. The method of embodiment 1A, further comprising:

[0303] At least two different oligonucleotide encoding molecules are separated by applying a voltage across the separation medium in a first direction.

[0304] Embodiment 8A. The method of embodiment 1A, further comprising:

[0305] A portion of the oligonucleotide encoding molecule is isolated by cutting the separation medium in a first cutting direction substantially perpendicular to the first direction to form separated fragments.

[0306] Embodiment 9A. The method of embodiment 8A, further comprising:

[0307] removing a portion of the oligonucleotide encoding molecule from the isolated fragment to form a restored portion of the oligonucleotide encoding molecule;

[0308] amplifying the oligonucleotide from the recovered portion of the oligonucleotide encoding molecule to form a portion of the copy sequence,

[0309] The copied sequence portion is sequenced to identify each coding region or combination of coding regions of the restored portion of the oligonucleotide encoding molecule to identify each positional building block of at least one encoded portion.

[0310] Embodiment 10A. The method of embodiment 7A, further comprising:

[0311] At least two different oligonucleotide encoding molecules are separated by applying a voltage across the separation medium in a second direction, wherein the second direction is substantially perpendicular to the first direction.

[0312] Embodiment 11A. The method of embodiment 10A, further comprising:

[0313] A portion of the oligonucleotide encoding molecule is isolated by cutting the separation medium in two directions, wherein the two directions are each independently substantially perpendicular to the first and second directions to form a rectangular separation fragment.

[0314] Embodiment 12A. The method of embodiment 11A, further comprising:

[0315] removing a portion of the oligonucleotide encoding molecule from the rectangular isolated fragment to form a restored portion of the oligonucleotide encoding molecule;

[0316] amplifying the oligonucleotide from the recovered portion of the oligonucleotide encoding molecule to form a portion of the copy sequence,

[0317] The copied sequence portion is sequenced to identify each coding region or combination of coding regions of the restored portion of the oligonucleotide encoding molecule to identify each positional building block of at least one encoded portion.

[0318] Embodiment 13A. The method of embodiment 1A, wherein the separation medium comprises at least one of particles and polymers, and the at least one target molecule is attached to at least one of the particles and the polymer.

[0319] Embodiment 14A. An electrophoresis system comprising:

[0320] A separation medium comprising:

[0321] Porous gel;

[0322] at least one target molecule within the porous gel; and

[0323] at least two different oligonucleotide encoding molecules within the porous gel,

[0324] wherein the oligonucleotide encoding molecule comprises an oligonucleotide linked to at least one encoded portion, wherein the oligonucleotide comprises at least two coding regions,

[0325] wherein at least one of the encoded portions contains at least two position building blocks,

[0326] Each position building block in each coded portion is identified by 2 to 5 coding regions.

[0327] Embodiment 15A. The electrophoresis system of embodiment 14A, wherein the porous gel further comprises:

[0328] At least one of a particle and a polymer, and at least one target molecule is attached to at least one of the particle and the polymer.

[0329] Computer Systems

[0330] The present disclosure provides computer systems programmed to implement the methods of the present disclosure, such as, for example, collecting target activity data for at least one resolved oligonucleotide-encoded molecule. Fig.12A computer system 1201 is shown, which includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1205, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1201 also includes a memory or storage location 1210 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1215 (e.g., a hard disk), a communication interface 1220 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1225, such as cache memory, other memory, data storage, and / or an electronic display adapter. The memory 1210, storage unit 1215, interface 1220, and peripheral devices 1225 communicate with the CPU 1205 via a communication bus (solid line) such as a motherboard. The storage unit 1215 can be a data storage unit (or data warehouse) for storing data. The computer system 1201 can be operably coupled to a computer network ("network") 1230 with the help of the communication interface 1220. The network 1230 may be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet that communicates with the Internet. In some cases, the network 1230 is a telecommunications and / or data network. The network 1230 may include one or more computer servers that may implement distributed computing, such as cloud computing. In some cases, with the help of the computer system 1201, the network 1230 may implement a peer-to-peer network that may enable a device coupled to the computer system 1201 to act as a client or server.

[0331] The CPU 1205 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a storage location, such as memory 1210. The instructions may be directed to the CPU 1205, which may then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 1205 may include fetching, decoding, executing, and writing back.

[0332] CPU 1205 may be part of a circuit, such as an integrated circuit. One or more other components of system 1201 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0333] Storage unit 1215 can store files, such as drivers, libraries, and saved programs. Storage unit 1215 can store user data, such as user preferences and user programs. In some cases, computer system 1201 may include one or more additional data storage units located external to computer system 1201, such as on a remote server that communicates with computer system 1201 via an intranet or the Internet.

[0334] Computer system 1201 can communicate with one or more remote computer systems via network 1230. For example, computer system 1201 can communicate with a user's remote computer system. Examples of remote computer systems include personal computers (e.g., portable PCs), touch-screen tablets or tablet computers (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or a personal digital assistant. A user can access the computer system 1201 through the network 1230.

[0335] The methods as described herein may be implemented by machine (e.g., computer processor) executable code stored on an electronic storage location (e.g., memory 1210 or electronic storage unit 1215) of computer system 1201. Machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by processor 1205. In some cases, the code may be retrieved from storage unit 1215 and stored in memory 1210 for ready access by processor 1205. In some cases, electronic storage unit 1215 may not be included, and machine executable instructions are stored on memory 1210.

[0336] The code may be precompiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language, which may be selected to enable the code to be executed in a precompiled or as-compiled manner.

[0337] Aspects of the systems and methods provided herein, such as computer system 1201, can be embodied in programming. Various aspects of the technology can be considered as "products" or "articles", which are usually present on a type of machine-readable medium or embodied in a type of machine-readable medium in the form of machine (or processor) executable code and / or related data. Machine executable code can be stored in an electronic storage unit, such as a memory (e.g., a read-only memory, a random access memory, a flash memory) or a hard disk. "Storage" type media may include any or all tangible memories of a computer, a processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transient storage for software programming at any time. All or part of the software can sometimes communicate via the Internet or various other telecommunications networks. For example, such communication can enable software to be loaded from a computer or processor to another computer or processor, such as from a management server or a host computer to a computer platform of an application server. Thus, another type of medium that can carry software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and through various air-links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered to be media that carry software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0338] Thus, machine-readable media such as computer executable code may take a variety of forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., such as can be used to implement the database shown in the accompanying drawings, etc. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and optical fiber, including the lines that constitute the bus within the computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: floppy disks, floppy disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card tapes, any other physical storage media with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cassette, a carrier wave that transmits data or instructions, a cable or link that transmits such a carrier wave, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0339] The computer system 1201 may include or communicate with an electronic display 1235 that includes a user interface (UI) 1240 for providing, for example, target activity data of at least one resolved oligonucleotide-encoded molecule. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0340] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1205. For example, the algorithm may implement a method for collecting target activity data of at least one resolved oligonucleotide-encoded molecule.

[0341] Various embodiments of the present disclosure are illustrated but not limited by the following examples. Those skilled in the art will recognize many equivalent techniques for performing the steps or portions of the steps recited herein. Example

[0342] Example 1. Preparation of synthetic compounds linked to DNA oligonucleotides

[0343] Those skilled in the art of DNA modification will appreciate that the reaction of coupling an activated acid with a reactive free amine can be used to rapidly and efficiently generate large quantities of compounds or a single compound.

[0344] Example 1A. Immobilize DNA on resin

[0345] To chemically modify a DNA oligonucleotide linked to a reactive amine handle, the DNA is first fixed to Add 40uL of 1:1 DEAE to each well of a 384-well filter plate (E&K Scientific, EK-2288). Storage solution.Hole is rinsed twice with 70uL water on plate vacuum manifold, then rinsed twice with 70uL binding buffer (10mM AcOH in distilled water).Plate is rotated 1 minute with 2000rpm in centrifuge to dry all liquids from hole.40uL binding buffer and 10uL 100ng / uL appropriate amine-connected DNA oligonucleotides are added to each hole, wide-mouthed pipette tip (Rainin) is used to mix (triturated) described hole, and incubated at room temperature for 10 minutes.Plate is rotated 1 minute with 2000RPM in receiving plate (greiner bio-oneREF 781201).The eluent in the collecting plate is added to the top of the resin, the described hole is mixed and incubated at room temperature for 5 minutes.Plate is rotated again in receiving plate (2000RPM 1 minute), and the DNA concentration of each eluent hole is analyzed by nanodrop to assess capture efficiency. If the measured DNA concentration is above 1 ng / uL, the eluate is added to the resin a third time and incubated for another 5 minutes and subjected to a long hold time (spin out), and then the measurement is repeated. If the measured DNA concentration is less than 1 ng / uL, the wells are rinsed on the vacuum manifold (3x70uL binding buffer, 3x70uL water, 3x70uL methanol).

[0346] Example 1B. General Procedure for Generation of Activated Acids and Coupling of DNA Oligonucleotides Linked to Reactive Amines

[0347] In order to couple the compound containing the acid moiety to the free amine of the DNA oligomer, the acid was activated in the following manner. For each acid, a 80:20DMF:MeOH solution with a concentration of 400mM was prepared as calculated based on the molecular weight of the acid. 1mL 40mM HoAT (5.5mg) solution was prepared in 80:20DMF:MeOH. Immediately before use, a stock solution of 100mM EDC*HCl 7.7mg was dissolved in 400uL MeOH. In order to generate the activated acid, 15uL HoAT (40mM) was added to 15uL of the desired acid (400mM), then 30uL DMF:MeOH 80:20, and finally 60uL EDC*HCl solution (100mM) was added, and the mixture was incubated at room temperature for 5 minutes.

[0348] Example 1C. General Procedure for Acylation of DNA with Activating Acids

[0349] In order to couple the activated acid solution with the DNA oligonucleotide connected to the reactive amine handle, the DNA fixed in the filter plate from Example 1A was rinsed 3 times with 70uL of 400mM DIPEA in DMF:MeOH 80:20 on the vacuum manifold, and then rinsed 3 times with 70uL MeOH. The filter plate was then placed on a rubber stopper to prevent the wells from draining. The required activated acid solution prepared in 70uL Example 1B was added to each well and the wells were mixed. The wells were sealed with metal tape (tape seal) (Corning, Cat.#6569) and incubated (RT, 1 hour). The plate was then unsealed and the solution was removed by a vacuum manifold. The bottom of the plate was sealed with a rubber stopper, and the activated acid of a fresh aliquot (70uL) was added to the appropriate wells, mixed and sealed with metal tape on the top of the plate and incubated (RT, 1 hour). After incubation, the plate seal was removed, and the solution was removed by a vacuum manifold and the wells were rinsed (3x, 70uL, 80:20DMF:MeOH).

[0350] Example 1D. Removal of the amine protecting group fluorenylmethoxycarbonyl (Fmoc)

[0351] For reactive amines attached to activated acid coupled and reactive DNA (Example 1C), for wells where the Fmoc protected acid was acylated, 60uL 4-methylpiperidine (20% v / v, DMF) was added and incubated for 10 minutes. The solution was removed by vacuum manifold and the wells were rinsed (3x70uL DMF, 3x70uL MeOH, 3x70uL DI water). The plate was rotated to remove any traces of liquid (1 minute, 2000RPM). This newly modified DNA oligonucleotide can be eluted (Example 1E) and additional couplings (as per Examples 1C-1D) can be performed to extend the compound.

[0352] Example 1E. From Elution, recovery and purification of synthetic modified oligonucleotides

[0353] In modification fixed After the reaction of the reactive amine that the DNA on the resin is connected, the DNA compound construct can be eluted from the hole into the collecting plate for analysis and purification. In the hole, 33uL elution buffer (distilled water of 1.5M NaCl, 50mM NaOH) is added, mixed and hatched for 5 minutes. The filter plate is placed on the receiving plate and collected by centrifugation (1 minute, 2000RPM). These steps are repeated twice to produce the DNA of elution in 99uL elution buffer. In the eluent collected, 2.5uL of 1M acetic acid (final concentration 25mM) is added to neutralize 50% NaOH, prevent the eluent from becoming acidic simultaneously. The sample is purified by Agilent 1200HPLC on Phenomenex Clarity 2.6um Oligo-XT 100A posts (50x2.1mm) using the HPLC method optimized for the specific compound-DNA construct of paying attention to.

[0354] Example 1F. Synthesis and production of full-length encoding positive control molecules appended to DNA

[0355] Those familiar with the art will appreciate that modified primers, such as DNA oligonucleotides modified with synthetic molecules from Examples 1A-1E, can be used to produce longer DNA constructs. The BCA positive control is synthesized on selected DNA oligonucleotides as described. The purified compound-DNA hybrid is used as a primer in standard PCR, using a full-length chain template specific to compound features. In brief, 10uM compound-Za-DNA hybrids of 1uL are added to the template strand composed of Za'-A(097-107)-Zbi-Bi-Zbf-Bf-Zci-Ci-Zcf-Cf-Zd-D001-Zf and enter the 25uL Q5 PCR reaction, and as will be appreciated by those skilled in the art, the optimized PCR program is run to generate "full length" 234 oligonucleotide codes, simulating the length and composition of the coding portion of the DNA encoding library. The standard protocol purification product of the thermoscientific GeneJet PCR purification kit is used.

[0356] Example 2 Fluorescent labeling of compounds on DNA oligonucleotides by orthogonal reaction conditions

[0357] Compounds of known binding affinity that are attached to DNA are produced and purified, wherein modified nucleotides are used to attach fluorescent compounds to visualize the DNA. Attachment of fluorophores (fluorescein in this example) is known in the art to enable determination of binding affinity and can also be used to visualize the location of a compound on a surface, gel, or sample.

[0358] 2A. Modified DNA Codons Using Commercially Available Alkyne-Modified Oligonucleotides

[0359] As is familiar to those skilled in the art, the use of an orthogonal reaction handle, in this case an alkyne, allows for synthetic modification of distal sites of the compound of interest. To produce fluorescent compounds, alkyne-modified DNA nucleotides (i5OctdU) oligonucleotides with reactive amine-linked ends (5AmMC6) were used to synthesize the desired compounds (as shown in Figure 6) and purified as in Examples 1B-1E. After purification, the sample was dried by speed-vac and dissolved in 20uL of DI water. To this solution was added 10uL of 7.3mM 5-FAM azide (Lumiprobe, Cat.#C4130), followed by 80uL of freshly prepared Click reaction buffer (120mM phosphate, 1.2mM aminoguanidine, 3mM tris(benzyltriazolylmethyl)amine, 6mM copper sulfate, 12mM sodium ascorbate in water) and incubated (30 minutes, room temperature). After incubation with 80uL Click quenching buffer (100mM Tris, 10mM EDTA, 0.005% Tween, 0.002% SDS in deionized water), 500uL binding buffer was added. 40uL DEAE resin was added to the Eppendorf tube solution and gently shaken for incubation (20 minutes, room temperature). The tube was centrifuged on a benchtop centrifuge for 1 minute and the supernatant was removed by pipetting. The resin was resuspended in 50uL water and transferred to a 384-well filter plate. The wells were rinsed on a vacuum manifold (3x70uL DI water, 3x70uL MeOH, 1x70uLDMSO, 3x70uL methanol, 3x70uL water). DNA was eluted using the above procedure (3x33uL elution buffer into a collection plate). The eluent was neutralized with 10uL of 100mM AcOH. The sample was purified using HPLC using standard methods.

[0360] Example 3 Using fluorescence polarization to measure the binding affinity of fluorescent compounds on DNA in solution to targets

[0361] 1. General Procedure for Fluorescence Polarization Using Soluble Protein as a Positive Control

[0362] Figure 6 shows the positive control compounds used.

[0363] Those skilled in the art will appreciate that, compared with unmodified natural compounds, the compound covalently attached to DNA may have modified, higher or lower or unchanged affinity characteristics. In order to solve this problem, using common binding assays, fluorescence polarization is used to evaluate selected positive control molecules, which are synthesized as tethered portions of DNA oligonucleotides that can be fluorescently labeled using orthogonal chemistry. The positive control compound on DNA (as shown in Figure 6) is diluted to a final concentration of 20nM in 1mL buffer in 1xTrisBorate buffer (45mM, pH 8.19) alone. This 20nM stock solution is diluted 1:1 to generate 10nM stock solution. 100uM BCA-II stock solution is prepared in 1x TrisBorate buffer. 10uL of 20nM stock solution of appropriate compounds is added to 384-well polystyrene F-type bottom small volume hibase non-binding black plates (Greinerbio-one, catalog number 784900) in row 1. Add 10uL of the appropriate compound 10nM stock solution in columns 2-20. Add 10uL of 100uM BCA-II protein solution to column 1, mix the wells and transfer 10uL of the wells to well column 2, and mix by pipetting, repeat the process until the 20th well. This procedure produces a reserve protein concentration diluted 1:1 in each subsequent well, thereby providing a range of 50uM to 95pM, while the concentration of the fluorescently labeled DNA compound remains constant at 10nM. Using 485 / 530 excitation / emission and pre-programmed fluorescence polarization methods, the plate is read by a Spectramax M5 plate reader. The values ​​obtained are plotted and the results are fitted to the Hill equation to produce binding affinity (Kd) recorded in nanomolar values.

[0364] Example 4 Generation of affinity electrophoresis retention lanes for selecting positive and negative trait molecules.

[0365] Example 4A: Efficient generation of target protein tags with capture moieties for immobilization

[0366] As will be appreciated by those skilled in the art, the modification of protein is widely used and commonplace, in this embodiment, the carbonic anhydrase isozyme II from bovine erythrocyte is modified with biotin to allow the target to capture the particle of streptavidin coating. In order to achieve this purpose, 25mg (0.833 micromole) carbonic anhydrase isozyme II from bovine erythrocyte (σC2552-25MG) is added PBS (pH 7.8) of 450uL. 1.5mg (2.5 micromole, 3 equivalents) NHS-dPeg4-biotin (Sigma QBD10200) dissolved in 550uL PBS (pH 7.8) is added to the solution. The mixture (800uM protein final concentration) is placed overnight at 4°C. The biotinylated protein can be used without purification in subsequent steps.

[0367] Example 4B: Generation of immobilized target on streptavidin-labeled agarose resin

[0368] Biotinylated target protein (bovine carbonic anhydrase isozyme II) is fixed on the resin of concern.To achieve this, 10.6mL 50% high capacity streptavidin agarose resin (Thermo#20361) slurry is added in 50mL falcon tube.By following procedure, resin is rinsed twice with triborate buffer (pH8.19): slurry is centrifuged 1 minute with 1000RPM, and supernatant is removed by pipette, 5mL triborate buffer is added thereto, and the procedure is repeated.After removing second washing liquid (wash), 1mL 800uM biotinylated bovine carbonic anhydrase prepared as above is added.By gentle stirring, mix this slurry and make it incubate at room temperature for 2 hours.After incubation, sample is centrifuged (1000RPM, 1 minute) and supernatant is carefully removed, while carefully allowing resin to keep "wetting".

[0369] Example 4C: Generation of immobilized target on optional streptavidin-labeled resin particles.

[0370] Those skilled in the art will recognize that the immobilization technique described in Example 4B can be easily modified to accommodate alternative resin types. In this example, streptavidin-coated silica particles (Sphero SVSIP-05-5 0.4-0.6um), streptavidin-coated polystyrene particles (Sphero SVP-05-10 0.4-0.69um) and streptavidin-coated agarose particles (Thermofisher 20347 with lower loading) were subjected to the same procedure as above to produce particles coated with target protein.

[0371] Example 4D uses low melting point agarose and target labeled particles to generate affinity electrophoresis resident agarose mixture.

[0372] In order to fix the target coated particles to the appropriate porous medium for electrophoresis, those skilled in the art will recognize that the temperature of sample preparation can be controlled to prevent the target from unfolding. In order to achieve this control and produce fixed target particles in the separation medium, low melting point agarose is used. 100mL of 0.5X tris borate buffer (pH 8.19) was added to a beaker containing 4 grams of UltraPure low melting point agarose (Invitrogen16520) to generate a 4% agarose solution. The beaker was heated in a high-speed microwave with brief stirring in between intervals of 1 minute until all the agarose was dissolved and minimal bubbles were observed. The dissolved gel was transferred to two 50mL falcon tubes, cooled and maintained at 42°C in a heating unit. A 2% low melting point agarose solution was prepared separately as described above using 2 grams of low melting point agarose and 100mL of 0.5× tris borate buffer (pH 8.19). 5.3mL sedimented high capacity streptavidin resin sample loaded with target protein was warmed to 42°C in a heating unit. 8mL of 4% low melting point agarose was added to the warm target particles at 42°C. The slurry was thoroughly mixed with a pipette tip while carefully avoiding bubbles. This mixture was able to generate 9 cm affinity electrophoresis resident lanes of 4 lanes using a custom-made electrophoresis mold (semi-cylinder, 4 mm high 8 mm cross section). It can be appreciated that the scale of this preparation can be adjusted to a lower or higher amount according to the availability of the target, the availability of the particles or the required number and length of lanes. The prepared 2% agarose solution was used to create a loading point for the sample in the agarose resident lane. In short, a loading comb (size as described below) was placed 1 cm from the top of the gel mold, and 2% agarose was added to surround the semi-cylinder loading point. The 2% agarose was allowed to solidify at room temperature and the loading comb was carefully removed from the gel, which created a depression in each lane capable of accommodating 15 uL of loading solution containing the library or desired samples therebetween.

[0373] The mold is in the free online software The design specifications are as follows: the bottom LxWxD19x9x1 depth, with walls (19x0.5x0.8 cm) bracketing the bottom on both sides. Six evenly spaced (0.5 cm intervals) semi-cylinders are printed on the bottom (see Figure 3, 302) with dimensions of 19x0.8x0.4 cm. As shown in the figure, the "load comb" is printed on the bottom with dimensions of 8.8x1x0.4 cm in length x width x depth. Six half cylinders of 0.3 cm in height and 0.6 cm in width are printed on the "load comb bottom", with a spacing of 0.6 cm between the half cylinders, and bracketing the half cylinders are two cubes (0.3x0.3x0.3x cm) to fit the edges of the pouring mold, forcing the comb to be located inside the prepared mold and the half cylinders to be centered inside the half tube. The UPS Store utilizes SE Plus 3D printer and commonly used ABS (acrylonitrile butadiene styrene) printing material for printing.

[0374] To generate the analytically selected molds for lane preparation and imaging, the 3D printed molds were first aerosolized Universal Mold Release was lightly sprayed to create an even light coating on the 3D printed mold. The end openings of the mold were sealed with masking tape and 50 (prepared according to the manufacturer's instructions, with a weight ratio of 1:2 A:B, mixed thoroughly and sonicated for 2 minutes to remove bubbles) was carefully poured to avoid bubbles and left overnight. After setting, the clear-flex50 mold was carefully removed to generate a semi-cylindrical lane that could be filled with agarose gel. The mold was thoroughly rinsed with water and 70% ethanol and used without further modification.

[0375] Analysis of selected gel lanes

[0376] Example 4E. Generation of full affinity electrophoresis dwell lanes with loading spots.

[0377] To create a full affinity electrophoresis resident lane construct, sample loading spots can be generated to allow the sample to be introduced into the porous agarose containing the resident particles loaded with the target. Using a custom printed loading spot comb, the previously prepared 2% low melting point agarose is poured into a mold sealed by a filter plug. The gel is allowed to cool to room temperature, the agarose solidifies and the comb is carefully removed. Check the loading wells for any inconsistencies or holes. Gently return the comb to the loading well area and remove the excess gel. Another fresh filter plug is placed at an appropriate distance from the edge of the loading area (approximately 1-9 cm or any custom distance), and the target-loaded particle / low melting point agarose mixture is moved into the lane and allowed to cool to room temperature and solidify to create a "capture zone" of the gel. The filter plug is removed and 2% LMP agarose is added to the end of the "capture zone" to fill the lane to the end, providing additional distance for the sample molecules to move after encountering the resident "capture zone".

[0378] Example 5. Fractionation and separation of known binding molecules attached to DNA according to affinity during electrophoresis:

[0379] Example 5A. Fluorescence positive control electrophoresis by target activity electrophoresis retention lane

[0380] a. Fig. 7A , Figure 7B and Fig. 8A -D

[0381] As will be appreciated by those skilled in the art of affinity chromatography, the resulting affinity electrophoresis retention lanes will serve as a grading of the resonance time of molecules that interact with the target to delay their movement through the gel during electrophoresis. As described above, gel lanes were generated with a target bovine carbonic anhydrase II capture region comprising a high-load streptavidin agarose and immobilized target. These lanes were loaded with 12 uL of a single positive control compound ( FIG. 6 ) or a mixture of fluorescently labeled positive controls ( FIG. 6 ) Fig. 8A , Figure 8B , Figure 8C , Fig.8D , Fig. 9 and Fig.10 ), with 200ng of each compound and 2uL of gel loading buffer. The gel was run at 40V for 18 hours and imaged periodically to follow the progress of the separation on a Bio-Rad Gel Dock EZ Imager using a blue plate. As can be seen in Figure 6, the retention factors (Rf's) generated by the single compound experiments correspond to the affinities of the synthetic modified oligonucleotides. Mixtures of these control molecules ( Fig. 8A -D, 9 and 10).

[0382] Example 5B. Fluorescence positive control electrophoresis by affinity electrophoresis retention lanes with different particle types and loading densities

[0383] a. Fig.10

[0384] The use of separation media with variable density and particle type provides an optional resident ability with enhanced characteristic potential. In order to evaluate this point, the swimming lane generated contains the following resident area with capture target position at 3cm: 1. polystyrene with streptavidin labeling for capturing target, 2. silica with streptavidin labeling for capturing target, 3. high load streptavidin agarose, 4. low load streptavidin agarose (wherein particle density equals 3), 5.1 / 4 density high load streptavidin agarose to swimming lane 3 (wherein total target amount equals 4). This explores particle type, surface display target density and target particle density. Use affinity to be 362nM (compound 10) and 7160nM (compound 5) compounds and fluorescein as tracer to experiment. Electrophoresis was carried out for 3 hours at 90V.

[0385] Example 5C. Full-length coding oligonucleotides and coding molecule isolation

[0386] The utility of this method is that it can separate trait positive compounds from trait negative compounds, and do so in such a way that trait positive compounds are graded by affinity for the target and sequenced to identify the coding region, thereby identifying the coding compound. Prior to electrophoresis, the gel apparatus was rinsed with de-DI water. The apparatus was filled with 2L of 0.5x TB buffer and placed in a deli fridge (below the gel apparatus line) in a water bath and cooled to 4°C. The gel mold with the lanes was loaded into the apparatus and cooled for 30 minutes. To the prepared resolution selection gel (using a 3 cm capture zone of high loading streptavidin agarose loaded with target protein) was added 12 uL of sample containing 2 uL of purple gel loading buffer (NEB # B7024S), 100 ng of the dummy library (consisting of 16 million unique library codes, but not synthetic modified compounds) and 50,000 copies of each positive control of bovine carbonic anhydrase II (Table XA), which were generated in Example 1F and independently and uniquely coded from the dummy library samples. The samples were electrophoresed at 90 V for 5 hours.

[0387] Example 5D Physical Distribution of Resident Media

[0388] Those familiar with the art will recognize that clean room technology is essential to prevent sample cross contamination. In order to achieve this after the gel is run, it is transferred to a laminar flow hood. The affinity selection gel is distributed into 44 1 mm slices and transferred to a sterile PCR plate. The slices are generated using a stack of 12 non-greased razor blades held between the index fingers and pressed against the gel slices. The PCR well position directly corresponds to the position of the gel slice.

[0389] Example 5E Recovery of DNA Oligonucleotides from Affinity Electrophoresis Retention Agarose Gel Lanes

[0390] In order to generate PCR copies of coding DNA from each assigned slice, DNA can be recovered in a form suitable for PCR amplification. To this end, the PCR plate containing the above-generated single gel slice is heated to 95°C for 5 minutes in a thermal cycler. The plate is centrifuged (2000RPM, 1 minute) and 3uL 10x B-Agarase I buffer and 5uL water containing 50,000 positive control DNA sequence molecules are added to each well to produce a total volume of 30uL. The plate is heated to 95°C again for 10 minutes and then placed in a heating unit at 42°C. The sample is kept on a 42°C heating unit for 10 minutes, and then 2uL B-agarase (New England BioLabs Cat#M0392L) is added to each well. The plate is then incubated (1 hour, 42°C). After incubation, the plate is centrifuged (2000RPM, 1 minute). If necessary, the plate can be sealed and stored at -20°C. The sample generated by this procedure is no longer able to gel, and can be amplified by PCR in subsequent steps.

[0391] Example 5F Indexing of individual gel slices by PCR installation of specific indexing oligonucleotide primers

[0392] In order to select the position of the DNA recovered by affinity selection gel identification, index oligonucleotide is installed on all amplification PCR copies of parental coding DNA chain. In order to realize this point from the gel sample of dissolution produced above, wells are mixed and 15uL is transferred to a new 96-well plate. Each gel slice is indexed separately by D096'-Zf index with zd'-D002'. Use Q5 High-Fidelity 2x Master Mix (NEB M0492L) to prepare a total of 5mL of the master mixture (mastermix). In each well, 1.25uL D-index primer (10uM) and 1.25uL Illumina-Za primer (10uM) are added, and 12 cycles are run using Illum_Za_T73 scheme.

[0393] Example 5G Post-amplification with index installation of Illumina Za and Illumina Zf primer sets for sequencing

[0394] In order to sequence the coding and index regions to properly assign the coding compound structure, a sequencing primer set can be installed. The Illumina primer set is installed using a standard PCR protocol. Briefly, 2uL is taken from each index-gel slice well and transferred to a new 96-well PCR plate. 23uL of Q5 master mix containing Illumina primer sets is added to these wells. The samples are run for 10 cycles using the Illum_Za-T73 protocol. After amplification, 2uL aliquots from each well are combined, and 1uL Exo-I (NEB M0293-L) is added to each 10uL combined PCR reaction. The combined samples are incubated at 37°C for 30 minutes and then heat-inactivated at 80°C for 15 minutes. The combined nuclease samples are purified using the GeneJet PCR Cleanup Kit (Thermo Cat. #K0701). The purity of the samples is assessed using 2% agarose gel and optical density measurement to quantify the amount of DNA. The purified DNA is submitted for NGS sequencing.

[0395] Example 5H Post-amplification with index installation of Illumina Za and Illumina Zf primer sets for sequencing

[0396] a. Fig.11A

[0397] The DNA samples prepared in Example 5F were submitted for DNA sequencing. Post-processing methods isolated and identified the A097-A107 coding region, which encodes the positive control molecule in the sample and the position index to determine the slice from which the code originated. Sequencing counts were plotted from the gel slice ( Fig.11A ), where compounds with the best affinities are highly retained compared to compounds with lower affinities and can be separated, sequenced, and identified.

Claims

1. A method for determining the relative target activity of at least one resolved oligonucleotide encoding molecule, comprising: providing a separation medium comprising a porous gel and a buffer system, wherein the separation medium comprises at least one target molecule attached to the separation medium; introducing a sample containing a mixture of at least a first oligonucleotide encoding molecule and a second oligonucleotide encoding molecule into a separation medium at a starting point, wherein the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule each include an encoding portion operably linked to an encoded portion; wherein the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule are different, and wherein the encoded portion of the first oligonucleotide encoding molecule and / or the second oligonucleotide encoding molecule is capable of interacting with a target molecule; forming at least a first resolved oligonucleotide encoding molecule and a second resolved oligonucleotide encoding molecule by separating the first oligonucleotide encoding molecule into a first location in a separation medium and separating the second oligonucleotide encoding molecule into a second location in the separation medium, wherein the separation is performed by electrophoresis; harvesting a first resolved oligonucleotide encoding molecule by fragmenting a first location in the separation medium to form a first resolved fragment, wherein the first resolved fragment includes the first resolved oligonucleotide encoding molecule; harvesting a second resolved oligonucleotide-encoded molecule by fragmenting a second location in the separation medium to form a second resolved fragment, the second resolved fragment comprising the second resolved oligonucleotide-encoded molecule; amplifying the coding portion of each first resolved oligonucleotide encoding molecule and the second resolved oligonucleotide encoding molecule by performing a polymerase chain reaction (PCR) on the coding portion of each first resolved oligonucleotide encoding molecule and the second resolved oligonucleotide encoding molecule to identify the encoded portion of each first resolved oligonucleotide encoding molecule and the second resolved oligonucleotide encoding molecule; and The relative target activity of the first resolved oligonucleotide encoding molecule is determined by correlating (1) the migration distance of the first resolved oligonucleotide encoding molecule from the starting point to the first position, and (2) the characteristics of the encoded portion of the first resolved oligonucleotide encoding molecule with (3) the migration distance of the second resolved oligonucleotide encoding molecule from the starting point to the second position.

2. The method according to claim 1, wherein the encoded portion of the first oligonucleotide encoding molecule and / or the second oligonucleotide encoding molecule is capable of binding to a target molecule.

3. The method of claim 1, wherein the at least one target molecule comprises at least one selected from the group consisting of: a cell, an oligonucleotide, a protein, an enzyme, a ribosome, and a nanodisc.

4. The method of claim 1, wherein the separation medium comprises at least one of the group consisting of: particles, polymers, and separation surfaces, and the at least one target molecule is attached to at least one of the particles, polymers, and separation surfaces. The method of claim 4 , wherein the particles comprise polymer particles or metal colloids.

6. The method of claim 4, wherein the polymer has a molecular weight of 10% or more of the lowest weight target molecule of the at least one target molecule.

7. The method of claim 1, further comprising separating the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule based on at least one target activity between at least one target molecule and the encoded portions of the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule.

8. The method of claim 7, wherein the at least one target activity comprises chemical modification of the encoded portion of the first oligonucleotide encoding molecule by the at least one target molecule.

9. The method according to claim 1, wherein: The coding portion contains at least two coding regions, and The encoded portion comprises at least two position building units, Each positional building block of the encoded portion is identified by 1 to 5 coding regions of the encoding portion.

10. The method according to claim 1, wherein: The first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule have a structure according to formula (I), (I)G-L-B in G is the coding portion of the coding region comprising at least two coding position building blocks; B is an encoded portion containing at least two position building units; L is a linker that covalently bonds G to B; and Each position building block in B is identified according to the position of 1 to 5 coding regions of G.

11. The method of claim 1, wherein the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule have a structure according to formula (II), (II)[(B1) M —L1] O —G—[L2—(B2) K ] P in G is the coding portion of the coding region comprising at least two coding position building blocks; B1 is a position building unit, and M represents an integer from 1 to 20; B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K; L1 is a linker that covalently bonds B1 to G; L2 is a linker that covalently bonds B2 to G; O is zero or 1; P is zero or 1; The condition is that at least one of O and P is 1; and Each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G.

12. The method of claim 1, wherein the first oligonucleotide encoding molecule and the second oligonucleotide encoding molecule have a structure according to formula (III), (III)[(B1) M —L1] O —G’—[L2—(B2) K ] P in G' comprises a coding portion, which includes the coding regions of at least two coding position building blocks and at least one hairpin region structure; B1 is a position building unit, and M represents an integer from 1 to 20; B2 is a position building unit, and K represents an integer from 1 to 20, wherein B1 is the same as or different from B2, and wherein M is the same as or different from K; L1 is a linker that covalently bonds B1 to G'; L2 is a linker that covalently bonds B2 to G'; O is an integer from 0 to 5; P is an integer from 0 to 5; Provided that at least one of O and P is an integer from 1 to 5; and Each position building block B1 at position M and / or B2 at position K is identified by 1 to 5 coding regions of G'.

13. The method according to claim 1, further comprising: by applying a first separation treatment in a first direction across the separation medium, separating the first oligonucleotide encoding molecule to a first location in the separation medium and separating the second oligonucleotide encoding molecule to a second location in the separation medium, The first separation process includes a first voltage scheme and a first duration.

14. The method according to claim 13, further comprising: harvesting the first resolved oligonucleotide-encoded molecule by segmenting a first location in the separation medium in a first segmentation direction substantially perpendicular to the first direction to form a first resolved fragment, the at least first resolved fragment comprising the first resolved oligonucleotide-encoded molecule; as well as The second resolved oligonucleotide-encoded molecule is harvested by segmenting the second location in the separation medium in a first segmentation direction substantially perpendicular to the first direction to form second resolved fragments comprising the second resolved oligonucleotide-encoded molecule.

15. The method according to claim 13, further comprising: by applying a second separation treatment across the separation medium in a second direction, separating the first oligonucleotide encoding molecule to a first location in the separation medium and separating the second oligonucleotide encoding molecule to a second location in the separation medium, wherein the second direction is substantially perpendicular to the first direction, The second separation process includes a second voltage scheme and a second duration.

16. The method according to claim 15, further comprising: harvesting the first resolved oligonucleotide-encoded molecule by segmenting the first position in the separation medium in a second segmenting direction substantially perpendicular to the first segmenting direction to form first resolved fragments, the first resolved fragments comprising the first resolved oligonucleotide-encoded molecule; and The second resolved oligonucleotide-encoded molecule is harvested by segmenting a second location in the separation medium in a second segmenting direction substantially perpendicular to the first segmenting direction to form second resolved fragments comprising the second resolved oligonucleotide-encoded molecule.

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