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WO 2016/123419 Al 4 August 2016 (04.08.2016) P O P C T

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WO 2016/123419 Al 4 August 2016 (04.08.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2016/123419 Al 4 August 2016 (04.08.2016) P O P C T (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, C12Q 1/68 (2006.01) C07H 21/02 (2006.01) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, PCT/US2016/015503 KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (22) International Filing Date: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 29 January 2016 (29.01 .2016) 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, (25) Filing Language: English TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 62/1 10,050 30 January 2015 (30.01.2015) US GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (71) Applicant: PRESIDENT AND FELLOWS OF HAR¬ TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, VARD COLLEGE [US/US]; 17 Quincy Street, Cam DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, bridge, MA 02138 (US). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors: SCHAUS, Thomas, E.; 90 Mount Vernon, Ar GW, KM, ML, MR, NE, SN, TD, TG). lington, MA 02476 (US). CHEN, Xi; 266 Melrose Street, Newton, MA 02466 (US). YIN, Peng; 51 Winthrop Road, Published: Brookline, MA 02445 (US). — with international search report (Art. 21(3)) (74) Agent: DIPIETRANTONIO, Heather, J.; Wolf, Green — before the expiration of the time limit for amending the field & Sacks, P.C., 600 Atlantic Avenue, Boston, MA claims and to be republished in the event of receipt of 02210-2206 (US). amendments (Rule 48.2(h)) (81) Designated States (unless otherwise indicated, for every — with sequence listing part of description (Rule 5.2(a)) kind of national protection available): AE, AG, AL, AM, (54) Title: MICROSCOPE-FREE IMAGING APR probes Full Record ! a,* polymerase \ (i) / polymerase ! i Half Record fl (ii) 4 stopper ig. 3 C (57) Abstract: Provided herein, in some aspects, are methods of imaging molecules without a microscope or other specialized equip ment, referred to herein as "microscope-free imaging (MFI)." Herein, "molecular instruments" (e.g., DNA-based and protein-based molecules) are used, instead of microscopes, in a "bottom-up" approach for inspecting molecular targets. MICROSCOPE-FREE IMAGING RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/1 10,050, filed January 30, 2015, which is incorporated by reference herein in its entirety. FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant Nos. 1DP2OD007292-01, 1R21HD072481-01 and 1R01EB018659-01, awarded by National Institutes of Health, Grant Nos. CCF 1162459, CCF 1054898 and CCF-13 17291, awarded by National Science Foundation, and Grant Nos. N00014-13-1-0593, N00014-14-1-0610 and N00014-1 1-1-0914, awarded by Office of Naval Research. The government has certain rights in the invention. BACKGROUND OF INVENTION Using a microscope to simultaneously image, at single molecule resolution, the relevant molecules of complex molecular systems of a living organism is challenging, particularly with respect to high-throughput and multiplexing analyses. Further, other techniques, such as co-immuno-precipitation and proximity ligation, often used to resolve molecular interactions, are inherently destructive, which prevents the repeat sampling required to elucidate individual molecular networks. SUMMARY OF INVENTION Provided herein, in some aspects, are methods of imaging molecules without a microscope or other specialized equipment. Such methods are referred to herein as "microscope-free imaging (MFI)" methods. Current imaging technologies use microscopes in a "top-down" approach to probe molecular targets. Herein, "molecular instruments" (e.g., DNA-based and protein-based molecules) are used, instead of microscopes, in a "bottom-up" approach for inspecting molecular targets. Methods of the present disclosure provide for higher spatial resolution (e.g., to visualize biological structures as well as true-connectivity and dynamics data within individual networks), greater multiplexing capabilities and higher throughput analyses relative to current microscopy imaging techniques. Further, methods of the present disclosure provide in situ access to molecular targets in their native states. Some aspects of the present disclosure provide nucleic acid barcoded probes that include a nucleic acid arranged to form a hairpin structure having a partially-double- stranded primer-binding region, a double-stranded barcode region, a double-stranded palindromic region, and a single-stranded loop region containing a target-binding moiety, wherein a synthetic non-DNA linker that terminates polymerization is located between the double- stranded palindromic region and the loop region. Some aspects of the present disclosure provide nucleic acid barcoded probes that include one or more nucleic acid strands arranged into (a) a double-stranded palindromic region, (b) a double-stranded barcode region, and (c) a primer-binding region. In some embodiments, a nucleic acid barcoded probe comprises a first nucleic acid strand comprising a first palindromic sequence, a first barcode sequence and a primer-binding sequence, and a second nucleic acid strand comprising a second palindromic sequence that is complementary to and binds to the first palindromic sequence and a second barcode sequence that is complementary to and binds to the first barcode sequence, wherein the first nucleic acid strand and the second nucleic acid strand are attached to each other through a linker (e.g., a non-nucleic acid linker, or a contiguous stretch of nucleic acids) and are arranged into a double-stranded palindromic region, a double-stranded barcode region, and a partially double stranded (or single-stranded) primer-binding region (e.g., forms a hairpin loop structure, such as the structure shown in Fig. 5). In some embodiments, a primer-binding region is single-stranded. In some embodiments, a primer-binding region is partially double-stranded. In some embodiments, a double-stranded palindromic region has a length of 4 to 10 nucleotide base pairs. In some embodiments, a double-stranded barcode region has a length of 2 to 100 nucleotide base pairs. In some embodiments, a primer-binding region has a length of 4 to 40 nucleotides. In some embodiments, a barcoded probe further comprises adjacent to the double- stranded palindromic region a molecule or modification that terminates polymerization. Two nucleic acid regions are considered "adjacent" to each other if they within 0 to 10 nucleotides of each other. In some embodiments, two regions are considered "immediately adjacent" to each other if there are no nucleotides between the two regions, e.g., the two regions are contiguous with one another. In some embodiments, a barcoded probe further comprises adjacent to the double- stranded palindromic region a synthetic non-DNA linker that terminates polymerization. For example, a barcoded probe may further comprise adjacent to the double-stranded palindromic region a triethylene glycol spacer that terminates polymerization. In some embodiments, a barcoded probe comprises a double-stranded displacement region adjacent to the molecule or modification that terminates polymerization. A double- stranded displacement region may have, for example, a length of 2 to 10 nucleotide base pairs. In some embodiments, a barcoded probe is arranged to form a hairpin structure comprising a single-stranded loop region. A single-stranded loop region may have, for example, a length of 3 to 50 nucleotides. In some embodiments, a barcoded probe further comprises a target-binding moiety. A target-binding moiety, in some embodiments, is attached to the single-stranded loop region. In some embodiments, a target-binding moiety is selected from the group consisting of: biotin (e.g., as a binding partner to avidin or streptavidin), an antibody (or an antibody fragment, e.g., Fc fragment), an aptamer, a nanobody, a nucleic acid, a drug and an atom. An "aptamer" is oligonucleic acid or peptide molecule that binds to a specific molecular target. A DNA, RNA or XNA aptamer typically includes a short (e.g., 5 to 30 nucleotides) strands of oligonucleotides. A peptide aptamer typically includes a short variable peptide domain, attached at both ends to a protein scaffold. A "nanobody" is a single-domain antibody (also referred to as antibody fragment) that includes a single monomeric variable antibody domain and is able to bind selectively to a molecular target. In some embodiments, a barcoded probe comprises at least one locked nucleic acid (LNA) nucleotide. A LNA may be located, for example, in or adjacent to the double- stranded barcoded region. In some embodiments, a barcoded probe further comprises a single-stranded poly-T (or poly-A) end sequence (e.g., at the 3' end or the 5' end). In some embodiments, a barcoded probe is bound to a molecular target through a target-binding moiety. Some aspects of the present disclosure provide pluralities of nucleic acid barcoded probes, as provided herein. In some embodiments, the double-stranded palindromic region is the same for each probe of the plurality. In some embodiments, the double-stranded barcode region is unique to each probe of the plurality.
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