Microscope-free imaging

Abstract

Provided herein, in some aspects, are methods of imaging molecules without a microscope or other specialized equipment, 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.

1 - 78 . (canceled) 
     
     
         79 . A method of detecting presence of a protein target, comprising:
 (a) providing, in a reaction mixture, the protein target, a strand-displacing polymerase, a plurality of first nucleic acid primers, a second nucleic acid primer, a first nucleic acid probe, and a second nucleic acid probe, wherein
 (i) the first nucleic acid probe and the second nucleic acid probe each comprises a molecule that terminates polymerization, a double-stranded unique molecular identifier (UMI) region comprising a UMI sequence, a double-stranded barcode region, and a single-stranded region, 
 (ii) the first nucleic acid probe is linked to a first antibody that specifically binds to a first epitope of the protein target, and the second nucleic acid probe is linked to a second antibody that specifically binds to a second epitope of the protein target, and 
 (iii) the first primers each comprises a single-stranded UMI region comprising a UMI sequence and specifically binds to the single-stranded region of the first nucleic acid probe, and the second primer specifically binds to the single-stranded region of the second nucleic acid probe; and 
   (b) incubating the reaction mixture under conditions that result in production of a plurality of nucleic acids, each comprising the UMI sequence of the first nucleic acid probe, the UMI sequence of the second nucleic acid probe, and the UMI sequence of one of the first nucleic acid primers; and   (c) sequencing the nucleic acids, wherein among the nucleic acids, the presence of different UMI sequences of the plurality of first nucleic acid primers but the same UMI sequence of the first nucleic acid probe and the same UMI sequence of the second nucleic acid probe, is indicative of the presence of the protein target.   
     
     
         80 . The method of  claim 79 , wherein each of the first and second nucleic acid probes forms a hairpin structure comprising a loop region adjacent to the molecule that terminates polymerization. 
     
     
         81 . The method of  claim 79 , wherein the conditions that result in the production of the nucleic acid comprise physiological conditions. 
     
     
         82 . The method of  claim 79 , wherein the conditions that result in the production of the nucleic acid comprise a temperature of 20-40 degrees Celsius, atmospheric pressure of 1, and/or a pH value of 6-8. 
     
     
         83 . The method of  claim 82 , wherein the conditions that result in the production of the nucleic acid comprise a temperature of 37 degrees Celsius and a period of time of 0.5 to 3.0 hours. 
     
     
         84 . The method of  claim 79 , wherein each of the double-stranded barcode regions has a length of 5 to 50 nucleotide base pairs, and/or wherein each of the single-stranded regions has a length of 4 to 50 nucleotides. 
     
     
         85 . The method of  claim 79 , wherein the molecule that terminates polymerization is a modified nucleotide, synthetic non-DNA linker, triethylene glycol spacer, or double-stranded displacement region. 
     
     
         86 . The method of  claim 79 , wherein the strand-displacing polymerase is a Bst large fragment polymerase, phi 29 polymerase, Deep VentR polymerase, Klenow fragment polymerase, or modified Taq polymerase. 
     
     
         87 . The method of  claim 79 , wherein, in the first nucleic acid probe or the second nucleic acid probe, the molecule that terminates polymerization is adjacent to the double-stranded barcode region, the double-stranded barcode region is adjacent to the double-stranded UMI region, the double-stranded UMI region is adjacent to the single-stranded region, and the a single-stranded region is located at a 3′ end of the first nucleic acid probe or the second nucleic acid probe. 
     
     
         88 . The method of  claim 79 , wherein each of the first and second nucleic acid probes comprise deoxyribonucleic acid. 
     
     
         89 . The method of  claim 79 , further comprising purifying the nucleic acids after (b). 
     
     
         90 . A composition comprising:
 a protein target, a strand-displacing polymerase, a plurality of first nucleic acid primers, a second nucleic acid primer, a first nucleic acid probe, and a second nucleic acid probe, wherein
 (i) the first nucleic acid probe and the second nucleic acid probe each comprises a molecule that terminates polymerization, a double-stranded unique molecular identifier (UMI) region comprising a UMI sequence, a double-stranded barcode region, and a single-stranded region, 
 (ii) the first nucleic acid probe is linked to a first antibody that specifically binds to a first epitope of the protein target, and the second nucleic acid probe is linked to a second antibody that specifically binds to a second epitope of the protein target, and 
 (iii) the first primers each comprises a single-stranded UMI region comprising a UMI sequence and specifically binds to the single-stranded region of the first nucleic acid probe, and the second primer specifically binds to the single-stranded region of the second nucleic acid probe. 
   
     
     
         91 . The composition of  claim 90  further comprising a plurality of nucleic acids, each comprising the UMI sequence of the first nucleic acid probe, the UMI sequence of the second nucleic acid probe, and the UMI sequence of one of the first nucleic acid primers. 
     
     
         92 . The composition of  claim 90 , wherein each of the double-stranded barcode regions has a length of 5 to 50 nucleotide base pairs, and/or wherein each of the single-stranded regions has a length of 4 to 50 nucleotides. 
     
     
         93 . The composition of  claim 90 , wherein the molecule that terminates polymerization is a modified nucleotide, synthetic non-DNA linker, triethylene glycol spacer, or double-stranded displacement region. 
     
     
         94 . The composition of  claim 90 , wherein the strand-displacing polymerase is a Bst large fragment polymerase, phi 29 polymerase, Deep VentR polymerase, Klenow fragment polymerase, or modified Taq polymerase. 
     
     
         95 . The composition of  claim 90 , wherein, in the first nucleic acid probe or the second nucleic acid probe, the molecule that terminates polymerization is adjacent to the double-stranded barcode region, the double-stranded barcode region is adjacent to the double-stranded UMI region, the double-stranded UMI region is adjacent to the single-stranded region, and the single-stranded region is located at a 3′ end of the first nucleic acid probe or the second nucleic acid probe.