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(12) Patent Application Publication (10) Pub. No.: US 2017/0137864 A1 Yin Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2017/0137864 A1 Yin Et Al US 2017.0137864A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0137864 A1 Yin et al. (43) Pub. Date: May 18, 2017 (54) HIGH-THROUGHPUT AND HIGHLY Related U.S. Application Data MULTIPLEXED IMAGING WITH (63) Continuation of application No. 15/108,911, filed on PROGRAMMABLE NUCLEC ACID PROBES Jun. 29, 2016, filed as application No. PCT/US 15/ (71) Applicant: President and Fellows of Harvard 20034 on Mar. 11, 2015. College, Cambridge, MA (US) (60) Provisional application No. 61/951.461, filed on Mar. 11, 2014. (72) Inventors: Peng Yin, Brookline, MA (US); Sarit Agasti, Jakkur (IN); Xi Chen, West Publication Classification Newton, MA (US); Ralf Jungmann, (51) Int. C. Munich (DE) CI2O I/68 (2006.01) (52) U.S. C. (73) Assignee: President and Fellows of Harvard CPC ......... CI2O 1/6804 (2013.01); C12O 1/6818 College, Cambridge, MA (US) (2013.01) (57) ABSTRACT (21) Appl. No.: 15/410,700 The present invention provides, inter alia, methods and compositions for imaging, at high spatial resolution, targets (22) Filed: Jan. 19, 2017 of interest. Patent Application Publication May 18, 2017. Sheet 1 of 7 US 2017/O137864 A1 Patent Application Publication May 18, 2017. Sheet 2 of 7 US 2017/O137864 A1 ~~~~~~); Patent Application Publication May 18, 2017. Sheet 3 of 7 US 2017/O137864 A1 Patent Application Publication May 18, 2017. Sheet 4 of 7 US 2017/O137864 A1 Patent Application Publication May 18, 2017. Sheet 5 of 7 US 2017/O137864 A1 Patent Application Publication May 18, 2017. Sheet 6 of 7 US 2017/O137864 A1 Patent Application Publication May 18, 2017. Sheet 7 of 7 US 2017/O137864 A1 1 O 3. O 2 n 10-1 O2 al a2 a3 a4 a5 a6 a7 as a9 a10 37°C, 150 mM (Nat), 0 mMMgt) E.23°C, 150 mM Nat), 0 m MMgt) 23 °C, 500 mM Nail, 10 mM Mg** FIG. 1 O US 2017/O 137864 A1 May 18, 2017 HIGH-THROUGHPUT AND HIGHLY imager Strands having an identical sequence may be iden MULTIPLEXED IMAGING WITH tically labeled. The first approach may be more convenient PROGRAMMABLE NUCLEC ACID PROBES as it requires a single excitation wavelength and detector. 0007. In this manner, it is possible to detect, image and/or RELATED APPLICATIONS quantitate two or more targets in a sample, regardless of their location in the sample, including regardless of whether their 0001. This application is a continuation of U.S. applica location in the sample is so close together to be indistin tion Ser. No. 15/108,911, filed Jun. 29, 2016, which is a guishable if signal from the two or more targets was national stage filing under 35 U.S.C. S371 of international observed simultaneously. Thus, the distance between two or application number PCT/US2015/020034, filed Mar. 11, more targets may be below the resolution distance of the 2015, which was published under PCT Article 21(2) in imaging system used to detect the targets, and still using the English and claims the benefit under 35 U.S.C. S 119(e) of methods provided herein it would be possible to distinguish U.S. provisional application Ser. No. 61/951.461, filed Mar. the two or more targets from each other, thereby facilitating 11, 2014, the entire contents of each of which are incorpo a more accurate and robust detection and quantitation of rated by reference herein. Such targets. In some instances, the resolution distance may GOVERNMENT SUPPORT be about 50 nm, as an example. 0008. It is to be understood that the “target content of a 0002 This invention was made with government support sample may be known or Suspected, or unknown and unsus under 1 DP2OD007292-01, 5R01EBO18659-02, and 1-UO1 pected, prior to performing the method. The binding partners MH106011-01 awarded by National Institutes of Health; contacting the sample may bind to the sample, or they may and under N00014-13-1-0593 awarded by U.S. Department not, depending on whether the target is present or absent of Defense Office of Naval Research; and under CCF (e.g., when the target is present, the binding partner may 1317291 awarded by National Science Foundation. The bind to the sample). The imager strands contacting the government has certain rights in the invention. sample may bind to the sample, or they may not, depending on whether the target is present or absent (e.g., when the FIELD OF THE INVENTION target is present, the imager strand may bind a corresponding docking Strand bound to the target). "Binding to the sample 0003. The invention relates generally to the field of means that the binding partner or the imager Strand is bound detection and quantification of analytes (e.g., targets). to its respective target or docking strand. BACKGROUND OF THE INVENTION 0009. The binding partners may be protein in nature, such as antibodies or antibody fragments. In the context of a 0004 Fluorescence microscopy is a powerful tool for binding partner that is an antibody or antibody fragment, the exploring molecules in, for example, a biological system. docking strands may be conjugated thereto at a constant However, the number of distinct species that can be distin region. The binding partner may be an antibody such as a guishably and simultaneously visualized (i.e. the multiplex monoclonal antibody, or it may be an antigen-binding anti ing power) is limited by the spectral overlap between the body fragment Such as an antigen-binding fragment from a fluorophores. monoclonal antibody. In some embodiments, the binding partner is a receptor. SUMMARY OF THE INVENTION 0010. The binding partner may be linked to the docking 0005. The present invention provides, interalia, methods Strand through an intermediate linker. In some embodiments, and compositions for detecting, imaging and/or quantitating an intermediate linker comprises biotin and/or streptavidin. targets (e.g., biomolecules) of interest. Some of the methods 0011. The imager strands may be fluorescently labeled provided herein involve (1) contacting a sample to be (i.e., they are conjugated to a fluorophore). Fluorophores analyzed (e.g., a sample Suspected of containing one or more conjugated to imager Strands of different nucleotide targets of interest) with moieties that bind specifically to the sequence may be identical to each other, or they may have targets (each moiety being a binding partner of a given an emission profile that overlaps or that doesn't overlap with target), wherein each moiety is conjugated to a nucleic acid that of other fluorophores. The fluorescently labeled imager (referred to herein as a docking strand) and wherein binding Strand may comprise at least one fluorophore. partners of different specificity are conjugated to different 0012. In some instances, fluorescently labeled imager docking strands, (2) optionally removing unbound binding nucleic acids such as imager Strands may comprise 1, 2, 3, partners, (3) contacting the sample with labeled (e.g., fluo or more fluorophores. rescently labeled) nucleic acids having a nucleotide 0013 The sample may be a cell, a population of cells, or sequence that is complementary to and thus specific for one a cell lysate from a cell or a population of cells. The target docking strand (such labeled nucleic acids referred to herein may be a protein. as labeled imager strands), (4) optionally removing unbound 0014. It will therefore be appreciated that the invention imager Strands, (5) imaging the sample in whole or in part provides a method for detecting analytes by binding analytes to detect the location and number of bound imager strands, to their respective binding partners and sequentially deter (6) extinguishing signal from the labeled imager Strand from mining the presence of Such binding partners, by repeatedly the sample (e.g., by bleaching, including photobleaching), binding, detecting and extinguishing (e.g., bleaching, Such and (7) repeating steps (3)-(6) each time with an imager as photobleaching) imager Strands, that optionally are iden Strand having a unique nucleotide sequence relative to all tically labeled (e.g., identically fluorescently labeled). other imager strands used in the method. 0015. Accordingly, the disclosure provides a method 0006 Imager strands may be identically labeled, includ comprising (1) contacting a sample being tested for the ing identically fluorescently labeled. In other embodiments, presence of one or more targets with one or more target US 2017/O 137864 A1 May 18, 2017 specific binding partners, wherein each target-specific bind 0025. The disclosure further provides a method compris ing partner is linked to a docking strand, and wherein ing (1) contacting a sample being tested for the presence of target-specific binding partners of different specificity are one or more targets with one or more target-specific binding linked to different docking Strands, (2) optionally removing partners, wherein each target-specific binding partner is unbound target-specific binding partners, (3) contacting the linked to a docking nucleic acid, and wherein target-specific sample with labeled imager strands having a nucleotide binding partners of different specificity are linked to differ sequence that is complementary to a docking Strand, (4) ent docking nucleic acids, (2) optionally removing unbound optionally removing unbound labeled imager Strands, (5) target-specific binding partners, (3) contacting the sample imaging the sample to detect location and number of bound labeled imager Strands, (6) extinguishing signal from the with labeled imager nucleic acids having a nucleotide bound labeled imager Strand, and (7) repeating steps (3)-(6), sequence that is complementary to a docking nucleic acid, each time with a labeled imager Strand having a unique (4) optionally removing unbound labeled imager nucleic nucleotide sequence relative to all other labeled imager acids, (5) imaging the sample to detect location and number Strands.
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