Directed protein evolution

Patent metadata

Field	Value
Publication number	US-2024003886-A1
Application number	US-202318335924-A
Country	US
Kind code	A1
Filing date	Jun 15, 2023
Priority date	Jun 15, 2022
Publication date	Jan 4, 2024
Grant date	—

Abstract

Aspects of the disclosure provide methods and compositions for determining the identity of an analyte using molecular barcodes and single-molecule directed evolution of target biomolecules (e.g., proteins or aptamers).

First claim

What is claimed is:

1 . A method comprising:
contacting an analyte with a first barcode recognition molecule and a second barcode recognition molecule, wherein the analyte is connected to a barcode comprising a first nucleic acid index sequence and a second nucleic acid index sequence, wherein the first barcode recognition molecule specifically binds to the first nucleic acid index sequence and the second barcode recognition molecule specifically binds to the second nucleic acid index sequence; detecting a series of signal pulses indicative of binding interactions between (i) the first barcode recognition molecule and the first nucleic acid index sequence, and (ii) the second barcode recognition molecule and the second nucleic acid index sequence; and determining the identity of the analyte based on the series of signal pulses.

2 . A method comprising:
contacting an analyte with a first barcode recognition molecule and a second barcode recognition molecule, wherein the analyte is connected to a barcode comprising a first nucleic acid index sequence, wherein the first barcode recognition molecule and the second barcode recognition molecule specifically bind to the first nucleic acid index sequence, wherein either (a) the first barcode recognition molecule binds to the first nucleic acid index sequence with a different affinity than the second barcode recognition molecule binds to the first nucleic acid index sequence or (b) the first barcode recognition molecule comprises a first detectable label and the second barcode recognition molecule comprises a second detectable label; detecting a series of signal pulses indicative of binding interactions between (i) the first barcode recognition molecule and the first nucleic acid index sequence, and (ii) the second barcode recognition molecule and the first nucleic acid index sequence; and determining the identity of the analyte based on the series of signal pulses.

3 . A method comprising:
(i) contacting an analyte with a first barcode recognition molecule and a second barcode recognition molecule, wherein the analyte is connected to a double-stranded barcode comprising a first nucleic acid index sequence and a second nucleic acid index sequence, wherein the first barcode recognition molecule specifically binds to the first nucleic acid index sequence and the second barcode recognition molecule specifically binds to the second nucleic acid index sequence; (ii) detecting a series of signal pulses indicative of binding interactions between (i) the first barcode recognition molecule and the first nucleic acid index sequence, and (ii) the second barcode recognition molecule and the second nucleic acid index sequence; and (iii) determining the identity of the analyte based on the series of signal pulses.

4 . A method comprising:
(i) contacting an analyte with a first barcode recognition molecule, wherein the analyte is connected to a double-stranded barcode comprising a first nucleic acid index sequence and a second nucleic acid index sequence, wherein the first barcode recognition molecule specifically binds to the first nucleic acid index sequence and specifically binds to the second nucleic acid index sequence; (ii) detecting a series of signal pulses indicative of binding interactions between (i) the first barcode recognition molecule and the first nucleic acid index sequence, and (ii) the first barcode recognition molecule and the second nucleic acid index sequence; and (iii) determining the identity of the analyte based on the series of signal pulses.

5 . The method of claim 3 or 4 , wherein, prior to (i), one or more segments of the nucleic acid strand that is bound to the index sequences are removed from the double-stranded barcode, optionally wherein this contacting step is performed in the presence of single-stranded binding (SSB) protein.

6 . The method of claim 5 , wherein the one or more segments of the nucleic acid strand that is bound to the index sequences are removed from the double-stranded barcode using incubation with enzymes or chemical means.

7 . The method of claim 3 or 4 , wherein, prior to (i), the double-stranded barcode is contacted with a nicking enzyme to remove one or more segments of the nucleic acid strands that is bound to the index sequences, optionally wherein the double-stranded barcode comprises restriction sites surrounding the index sequences that are recognized by the nicking enzyme, optionally wherein this contacting step is performed in the presence of single-stranded binding (SSB) protein.

8 . The method of any one of claims 5 - 7 , wherein the SSB protein is a herpes simplex virus (HSV-1) single-strand DNA-binding protein, a bacterial SSB, replication protein A, or Eukaryotic mitochondrial SSB.

9 . The method of any one of the preceding claims, wherein the analyte is a DNA molecule, an RNA molecule, a polypeptide, a protein, or a nucleic acid aptamer.

10 . The method of any one of the preceding claims, wherein the first nucleic acid index sequence and the second nucleic acid index sequence comprise different nucleotide sequences and/or different nucleic acid modifications.

11 . The method of any one of the preceding claims, wherein the first nucleic acid index sequence and/or the second nucleic acid index sequence comprises a length of 4-15 nucleotides, 5-10 nucleotides, 6-9 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, or 9 nucleotides.

12 . The method of any one of the preceding claims, wherein the nucleotide sequence of the second nucleic acid index sequence comprises one nucleobase substitution relative to the nucleotide sequence of the first nucleic acid index sequence.

13 . The method of any one of the preceding claims, wherein the barcode comprises a spacer between the first nucleic acid index sequence and the second nucleic acid index sequence.

14 . The method of claim 13 , wherein the spacer is a non-nucleic acid spacer or a nucleic acid spacer, optionally wherein the non-nucleic acid spacer is a polyethylene glycol spacer.

15 . The method of claim 14 , wherein the nucleic acid spacer comprises a length of 5-35 nucleotides, 10-25 nucleotides, 15-30 nucleotides, 10-20 nucleotides, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides.

16 . The method of any one of the preceding claims, wherein the barcode further comprises a third nucleic acid index sequence, optionally wherein the third nucleic acid index sequence comprises two nucleobase substitutions relative to the nucleotide sequence of the first nucleic acid index sequence.

17 . The method of claim 16 , wherein the first barcode recognition molecule specifically binds to the third nucleic acid index sequence.

18 . The method of claim 16 , wherein the analyte is contacted with a third barcode recognition molecule, wherein the third barcode recognition molecule specifically binds to the third nucleic acid index sequence.

19 . The method of any one of claims 16 - 18 , wherein the barcode further comprises a fourth nucleic acid index sequence, optionally wherein the fourth nucleic acid index sequence comprises three nucleobase substitutions relative to the nucleotide sequence of the first nucleic acid index sequence.

20 . The method of claim 19 , wherein the first barcode recognition molecule specifically binds to the fourth nucleic acid index sequence.

21 . The method of claim 19 , wherein the analyte is contacted with a fourth barcode recognition molecule, wherein the fourth barcode recognition mole

Assignees

• Quantum Si Inc
• Ribezzi Crivellari Marco
• Griffiths Andrew
• Pichard Kostuch Adeline
• Flamm Andrea

Inventors

• Ribezzi-Crivellari Marco
• Griffiths Andrew
• Pichard-Kostuch Adeline
• Flamm Andrea
• Spenkelink Lisanne
• Van Oijen Antonius M

Classifications

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• G01N2333/91245

  Nucleotidyltransferases (2.7.7) · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/6854

  Immunoglobulins · CPC title