TETHERED AMPLIFICATION PRODUCTS IN CELLS AND TISSUES

1 . A method of detecting a nucleic acid molecule in situ, said method comprising:

(i) extending an oligonucleotide hybridized to a circular polynucleotide to form an extended oligonucleotide, wherein the oligonucleotide is in a cell or tissue;

(ii) hybridizing a first sequence of a staple oligonucleotide to the extended oligonucleotide and hybridizing a second sequence of the staple oligonucleotide to the extended oligonucleotide; and

(iii) binding a primer to the extended oligonucleotide and incorporating with a polymerase one or more fluorescently labeled nucleotides into the primer and detecting the one or more incorporated fluorescently labeled nucleotides.

2 . The method of claim 1 , wherein the staple oligonucleotide comprises, from 5′ to 3′, the first sequence, a joining sequence, and the second sequence.

3 . The method of claim 2 , wherein the first sequence and the second sequence comprise the same sequence.

4 . The method of claim 2 , wherein the staple oligonucleotide comprises a 3′ phosphorothioate, 3′ C3 spacer nucleotide, 3′ reversible terminator, 3′ inverted nucleotide, 3′ abasic nucleotide, 3′ Locked Nucleic Acid (LNA) nucleotide, or a 2′,3′-dideoxynucleotide (ddNTP).

5 . The method of claim 1 , wherein the staple oligonucleotide includes a bioconjugate reactive moiety at a 5′ end.

6 . The method of claim 1 , wherein the staple oligonucleotide includes a 5′ biotin moiety, 5′ thio moiety, 5′ aldehyde moiety, or 5′ amine moiety.

7 . The method of claim 1 , wherein the staple oligonucleotide comprises a 5′ amine moiety.

8 . The method of claim 1 , wherein the staple oligonucleotide includes a 5-ethynyluridine nucleotide, 5-ethynylcytidine nucleotide, 8-azidoadenine nucleotide, 5-azidomethyluridine nucleotide, or 5-azido-2′-deoxyuridine (5-AzidodU) nucleotide.

9 . The method of claim 1 , further comprising contacting the staple oligonucleotide with a crosslinking molecule and binding the staple oligonucleotide to a cellular component.

10 . The method of claim 1 , further comprising attaching the staple oligonucleotide to a cellular component, wherein said attaching comprises forming a first bioconjugate linker between the crosslinking molecule and the staple oligonucleotide and forming a second bioconjugate linker between the crosslinking molecule and the cellular component.

11 . The method of claim 10 , wherein the crosslinking molecule is bis(sulfosuccinimidyl)suberate (BS3), (PEGylated bis(sulfosuccinimidyl)suberate) (BS(PEG)9, Mal-PEG-NHS ester, ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester), 1,6-hexanediol, 3-Maleimidobenzoic acid N-hydroxysuccinimide ester (MBS), 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester (Sulfo-SMCC), 4-(N-Maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), suberic acid bis(N-hydroxysuccinimide ester), adipic acid dihydrazide, dimethyl pimelimidate dihydrochloride (DMP), sulfo-dibenzocyclooctyne-biotin (Sulfo-DBCO-biotin), dibenzocyclooctyne-maleimide, Dibenzocyclooctyne-PEG-maleimide, 6-Maleimidocaproic acid N-succinimidyl ester, disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), sulfosuccinimidyl 4,4′-azipentanoate (Sulfo-SDA), sulfosuccinimidyl 6-(4,4′-azipentanamido)hexanoate (Sulfo-LC-SDA), succinimidyl 4,4′-azipentanoate (SDA), or succinimidyl 6-(4,4′-azipentanamido)hexanoate (NHS-LC-Diazirine).

12 . The method of claim 1 , wherein the circular polynucleotide comprises a target sequence and/or a barcode sequence.

13 . The method of claim 1 , prior to step (i), the method comprises:

contacting the cell or tissue with a polynucleotide probe and hybridizing a first hybridization sequence of the polynucleotide probe to a first target sequence of a nucleic acid molecule, and hybridizing a second hybridization sequence of the polynucleotide probe to a second target sequence of the nucleic acid molecule, wherein said nucleic acid molecule comprises a target sequence between the first target sequence and the second target sequence;

extending the polynucleotide probe along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming the circular polynucleotide; and

binding the oligonucleotide to the circular polynucleotide.

14 . The method of claim 1 , wherein the oligonucleotide is covalently attached to a protein-specific binding agent, wherein the protein-specific binding agent is an antibody, single-chain Fv fragment (scFv), affimer, aptamer, single-domain antibody (sdAb), or antibody fragment-antigen binding (Fab).

15 . The method of claim 1 , wherein the antibody or sdAb is bound to HLA-DR, ATPase, Ki67, CD45-RA, CD3, CD4, CD8, FOXP3, TIM-3, PD-1, CTLA-4, Ki67, IFNG, IL-10, IL-17, LAG-3, TIGIT, CD40, GITR, ICOS, OX40, CD25, KLRG1, CD27, CCR7, CXCR5, CD127, or CD39 in the cell or tissue.

16 . The method of claim 1 , prior to (i), the method comprises:

contacting the cell or tissue comprising the oligonucleotide with a polynucleotide comprising a first target hybridization sequence and a second target hybridization sequence;

hybridizing the first target hybridization sequence to the nucleic acid molecule and hybridizing the second target hybridization sequence to the oligonucleotide; and

ligating the first target hybridization sequence to the second target hybridization sequence to form the circular polynucleotide.

17 . The method of claim 1 , prior to (i), the method comprises:

contacting the cell or tissue comprising a nucleic acid molecule with a polynucleotide comprising a first target hybridization sequence and a second target hybridization sequence;

hybridizing the first target hybridization sequence to the nucleic acid molecule and hybridizing the second target hybridization sequence to the nucleic acid molecule; and

ligating the first target hybridization sequence to the second target hybridization sequence to form the circular polynucleotide; and binding the oligonucleotide to the circular polynucleotide.

18 . The method of claim 1 , wherein (i) and (ii) occur at different temperatures.

19 . The method of claim 1 , wherein (i) occurs at a first temperature, and step (ii) occurs at a second temperature, wherein the second temperature is greater than the first temperature.

20 . The method of claim 1 , wherein (ii) occurs after (i).