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(51) International Patent Classification: MC, MK, MT, NL, NO, PL, PT, RO

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(51) International Patent Classification: MC, MK, MT, NL, NO, PL, PT, RO ( (51) International Patent Classification: MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, C12Q 1/6811 (2018.01) C40B 40/06 (2006.01) TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, C12Q 1/6853 (2018.01) C40B 40/08 (2006.01) KM, ML, MR, NE, SN, TD, TG). C12Q 1/6855 (2018.01) Published: (21) International Application Number: — with international search report (Art. 21(3)) PCT/US20 19/035481 — with sequence listing part of description (Rule 5.2(a)) (22) International Filing Date: 05 June 2019 (05.06.2019) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/680,946 05 June 2018 (05.06.2018) US (71) Applicant: ARRAKIS THERAPEUTICS, INC. [US/US]; 830 Winter Street, Waltham, Massachusetts 0245 1 (US). (72) Inventors: BLAIN, Jonathan Craig; 148 Boston Rock Road, Melrose, Massachusetts 02176 (US). BARSOUM, James Gregory; 6 Moreland Avenue, Lexington, Massa¬ chusetts 02421 (US). KUBICA, Neil; 57 Lincoln House Point, Swampscott, Massachusetts 01907 (US). PETTER, Jennifer C.; 22 Robinwood Land, Stow, Massachusetts 01775 (US). SELETSKY, Alexandra East; 60 Quincy Street, Medford, Massachusetts 02155 (US). (74) Agent: REID, Andrea L.C. et al.; One International Place, 40th Floor, 100 Oliver Street, Boston, Massachusetts 021 10-2605 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW,KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available) : ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (54) Title: ENCODED LIBRARIES AND METHODS OF USE FOR SCREENING NUCLEIC ACID TARGETS (57) Abstract: The present invention provides encoded libraries of small molecules that may be used to screen for binding to a nucleic acid target, such as an RNA or relevant fragment thereof implicated in a disease, disorder, or condition. The present invention also provides enriched encoded libraries and methods for preparing the same. The present invention further provides methods and kits for ligation, such as proximity -based ligation, of the nucleic acid target to a nucleic acid that encodes a small molecule library member, thus enabling methods of screening, preparation of enriched libraries, identification of screening hits, and sample processing. ENCODED LIBRARIES AND METHODS OF USE FOR SCREENING NUCLEIC ACID TARGETS TECHNICAL FIELD OF THE INVENTION [0001] The present invention relates to encoded libraries and methods of use thereof for screening and identifying candidate compounds for binding to a nucleic acid target of interest. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of U.S. Provisional Application No. 62/680,946, filed on June 5, 2018, the entirety of which is hereby incorporated by reference. SEQUENCE LISTING [0003] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 3, 2019, is named 394457-005WO_l67379_SL_ST25.TXT and is 3,642 bytes in size. BACKGROUND OF THE INVENTION [0004] Ribonucleic acids (RNAs) historically have been considered mere transient intermediaries between genes and proteins, whereby a protein-coding section of deoxyribonucleic acid (DNA) is transcribed into ribonucleic acid (RNA) that is then translated into a protein. Most RNA was thought to lack defined tertiary structure, and even where tertiary structure was present it was believed to be largely irrelevant to the RNA’s function as a transient messenger. This understanding has been challenged by the recognition that RNA, including non-coding RNA (ncRNA), plays a multitude of critical regulatory roles in the cell and that RNA can have complex, defined, and functionally essential tertiary structure. [0005] Intervention in the transcriptome has the potential to treat a vast array of diseases, but has mostly been investigated with nucleic acid-based therapeutic modalities such as antisense RNA or siRNA. Unfortunately, in most cases such approaches have yet to overcome significant challenges including drug delivery, absorption, distribution to target organs, pharmacokinetics, and cell penetration. In contrast, small molecules have a long history of successfully surmounting these barriers and these qualities, which make them suitable as drugs, and they are readily optimized. However, there are no validated, general methods of screening small molecules for binding to RNA or other nucleic acid targets in general. Consequently, there exists an urgent need for new methods to screen libraries of small molecules against a nucleic acid target of interest or even a library of nucleic acid targets. [0006] DNA-encoded chemical libraries (DEL) are a technology that enables the synthesis and screening, on a massive scale, of libraries of small molecules. DEL technology bridges the fields of combinatorial chemistry and molecular biology and represents a well-validated tool for drug discovery against protein targets. The aim of DEL technology is to enable massive parallel screening in early phase drug discovery efforts such as target validation and hit identification, thereby accelerating and decreasing costs in the drug discovery process. [0007] DEL technology generally uses DNA “barcodes” to give each library member a unique identifier. In some cases the DNA sequences include segments that direct and control chemical synthesis of small molecule library members from building block precursors. The technique enables massively parallel creation and interrogation of libraries via affinity selection, typically on an immobilized protein target. Homogeneous methods for screening DNA-encoded libraries are also available using, for example, water-in-oil emulsion technology to isolate individual ligand- target complexes that are later identified. [0008] Current DEL technologies evaluate test compound binding against a protein target that is in solution, immobilized in a matrix, or is conjugated to a DNA barcode. However, a validated approach to DEL library screening against a nucleic acid target does not exist. Thus, current DEL technologies have not been shown to allow screening libraries of small molecules against targets that are themselves nucleic acids. The ability to screen compound libraries against a nucleic acid target would enable medicinal intervention in the transcriptome. Thus, there is a need in the art for improved DEL technologies capable of being applied to one or more nucleic acid targets. BRIEF DESCRIPTION OF THE FIGURES [0009] FIG. 1 shows cartoons of how a YoctoReactor® (yR) is used to prepare a DEL. (a) Self-assembly of chemical building blocks (BBs) conjugated to conserved DNA sequences serve to direct the synthesis and simultaneously record the synthetic route via a distal BB coding region. The conserved DNA is designed to self-assemble into a three-way junction (3WJ) or four-way DNA junction (4WJ), thus three or four BBs can be brought into close proximity and allowed to react (b) Representation of an arm of the yR. BBs are attached via cleavable or non-cleavable linkers to bispecific DNA oligonucleotides (oligo-BBs) designed to contain a DNA barcode for the attached BB at the distal end of the oligo and an area of conserved DNA sequence that self- assembles the DNA into a 3WJ or 4WJ. (c) Two reactants are brought into close proximity at the cavity at the center of a yR DNA junction, which has a volume of about one yoctoliter (10 24 L). The close proximity of the two reactants at the cavity results in a high effective concentration of the reactants and facilitates high reaction rates between the BB and acceptor (d) Representative member of a DEL library prepared from a yR comprising a 3WJ. The size of a DEL library is determined by the number of different BB-oligos as well as yR geometry. [0010] FIG. 2 shows a cartoon scheme for preparing a small molecule DEL library member (display product) by the yR approach. Each DNA strand contains a codon region which encodes for the particular BB. In the first step, repertoires of two DNA strands with individual codons and BB conjugates are mixed together with a complementary DNA strand that assembles the yR. Because of sequence complementarity, these DNA strands self-assemble combinatorially into a stable three-way junction forming the stable double-stranded framework of the yR. The BBs are then coupled in a chemical reaction. Repetition with a third BB-oligo and cleavage of all by one of the BB linkers followed by purification and primer extension leads to the library member. [0011] FIG. 3 shows a scheme of the Binder Trap Enrichment® (BTE) method of library screening. A DEL mixed with a DNA-labeled target is allowed to reach equilibrium in solution where the target concentration can be controlled. A rapid dilution during which the binding kinetics become dominated by dissociation is followed by a rapid emulsion formation which traps the bound ligands with the target within aqueous emulsion droplets. Once trapped, the target and the library DNA are ligated inside the droplets.
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