Detection of analytes in cells and tissues

A system for detecting biomolecules in a cell or tissue, the system comprising:

• a substrate comprising a channel, wherein the cell or tissue is immobilized within the channel;
• a first oligonucleotide comprising a cleavable site, wherein cleaving the first oligonucleotide at the cleavable site produces a first fragment and a second fragment, each having a melting temperature lower than a melting temperature of the first oligonucleotide;
• a second oligonucleotide;
• a cleaving agent configured to cleave the cleavable site of the first oligonucleotide;
• a polymerase;
• and
• a plurality of labeled nucleotides,
• wherein the system is configured to sequentially:
• (i) hybridize the first oligonucleotide to a first target polynucleotide in the cell or tissue and detect a signal associated with the first oligonucleotide, (ii) cleave the first oligonucleotide at the cleavable site and remove the first fragment and the second fragment, and (iii) hybridize the second oligonucleotide to a second target polynucleotide in the cell or tissue and detect a signal associated with the second oligonucleotide.

A method of detecting a plurality of proteins in a cell or tissue, the method comprising:

• (a) contacting the cell or tissue with a plurality of circularizable probes and forming a plurality of oligonucleotide complexes, wherein the cell or tissue comprises the plurality of proteins and each protein comprises an oligonucleotide, and forming an oligonucleotide complex comprises binding a circularizable probe to the oligonucleotide;
• (b) circularizing each circularizable probe to form a plurality of circular polynucleotides, wherein each circular polynucleotide comprising a primer binding sequence and a barcode sequence;
• (c) amplifying the circular polynucleotides by rolling circle amplification to generate a plurality of amplification products;
• (d) hybridizing a first sequencing primer to a first subset of the amplification products, wherein the first sequencing primer comprises a cleavable site, incorporating with a polymerase a first labeled nucleotide into the first sequencing primer, and detecting the first labeled nucleotide to identify a first subset of target proteins;
• (e) cleaving the first sequencing primer at the cleavable site to produce a first fragment and a second fragment, and removing the first fragment and the second fragment;
• and
• (f) hybridizing a second sequencing primer to a second subset of the amplification products, incorporating with a polymerase a second labeled nucleotide into the second sequencing primer, and detecting the second labeled nucleotide to identify a second subset of target proteins.