OLIGONUCLEOTIDE CONJUGATES USEFUL FOR IN SITU TARGET DETECTION
Disclosed herein, inter alia, are compositions and methods useful for interrogating a cell and/or tissue.
1 . A method of detecting a target molecule in or on a cell or tissue, said method comprising
binding a specific binding agent comprising an oligonucleotide to the target molecule in or on a cell or tissue, wherein said oligonucleotide comprises a first blocking oligonucleotide hybridized to a first sequence of the oligonucleotide, and a second blocking oligonucleotide hybridized to a second sequence of the oligonucleotide;
removing said blocking oligonucleotides and binding a polynucleotide probe comprising a first binding sequence and a second binding sequence to the oligonucleotide;
amplifying the polynucleotide probe to form an amplification product comprising one or more copies of the first binding sequence and the second binding sequence; and
binding a fluorescent moiety to the amplification product and detecting the fluorescent moiety, thereby detecting the target molecule.
2 . The method of claim 1 , wherein the specific binding agent is an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, or an aptamer.
3 . The method of claim 1 , wherein the specific binding agent is an antibody, single-chain Fv fragment (scFv), or antibody fragment-antigen binding (Fab).
4 . The method of claim 1 , wherein the specific binding agent is an enzyme, enzyme mutant, peptide, Molecular Imprinted Polymer (MIP), DARPin (Designed Ankyrin Repeat Protein), peptoid, lectin, siRNA, or miRNA molecule.
5 . The method of claim 1 , wherein binding the specific binding agent comprises incubation in a buffer at 30° C. to 40° C.
6 . The method of claim 5 , wherein binding the polynucleotide probe comprises incubation in a buffer at 40° C. to 50° C.
7 . The method of claim 6 , wherein amplifying comprises incubation in a buffer at 30° C. to 40° C.
8 . The method of claim 7 , wherein amplifying comprises rolling circle amplification for 15 minutes to 16 hours.
9 . The method of claim 7 , wherein amplifying comprises rolling circle amplification for 15 minutes to 1 hour.
10 . The method of claim 1 , wherein the fluorescent moiety comprises a fluorescently labeled oligonucleotide.
11 . The method of claim 1 , wherein the fluorescent moiety is a fluorescently labeled nucleotide, and the method further comprises binding a sequencing primer to the amplification product and binding the fluorescently labeled nucleotide to the sequencing primer.
12 . The method of claim 1 , wherein the oligonucleotide is 30 to 40 nucleotides.
13 . The method of claim 1 , wherein the target molecule is selected from: PD-L1, CD8, CD3, PD-1, CD45, CD4, CD68, CD11c, FoxP3, α-SMA, CD20, Ki67, CD56, CD31, CTLA-4, PanCK, CD45RO, CD45RA, ATPase, Pan-Cadherin, Vimentin, Beta-2-microglobulin, and HLA-DR.
14 . A computer-implemented method for designing oligonucleotide sequences, said method comprising:
generating a plurality of oligonucleotide sequences comprising 20 to 40 nucleotides;
removing oligonucleotide sequences comprising a GC percentage greater than 60% over a portion of the oligonucleotide sequence;
removing oligonucleotide sequences comprising five consecutive strong bases and or five consecutive weak bases;
removing oligonucleotide sequences comprising a secondary structure; and.
removing oligonucleotide sequences comprising complementarity to the transcriptome.
15 . The computer-implemented method of claim 14 , further comprising removing oligonucleotide sequences comprising complementarity to the genome.
16 . The computer-implemented method of claim 14 , further comprising removing oligonucleotide sequences with a homopolymer sequence greater than 4 nucleotides.
17 . An oligonucleotide comprising a sequence formed according to the method of claim 14 .
18 . The oligonucleotide of claim 17 , wherein said oligonucleotide is covalently attached to a specific binding agent, wherein the specific binding agent is an antibody, single-chain Fv fragment (scFv), antibody fragment-antigen binding (Fab), affimer, aptamer, enzyme, peptide, Molecular Imprinted Polymer (MIP), DARPin (Designed Ankyrin Repeat Protein), peptoid, or lectin.
19 . A cell or tissue comprising the oligonucleotide of claim 18 .
20 . A composition comprising:
a specific binding agent covalently attached to an oligonucleotide, wherein
the oligonucleotide is hybridized to a first blocking oligonucleotide and a second blocking oligonucleotide,
the oligonucleotide is 30 to 40 nucleotides
the oligonucleotide does not include five consecutive weak bases and/or five consecutive strong bases; and
the oligonucleotide does not comprise secondary structure.