# US 11,906,525 B2 · Single molecule peptide sequencing methods

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- Number: US 11,906,525 B2
- Document ID: US11906525B2
- Status: Granted
- Kind: B2
- Application number: 17/824,073
- Filed: 2022-05-25
- Published: 2022-09-15
- Granted: 2024-02-20
- Earliest claimed: 2019-06-20
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Edward Boyden; Adam Henry Marblestone; Samuel Gordon Rodriques
- Web page: https://patentgrep.com/patent/US11906525B2

## CPC

- G01N 33/6824: {involving N-terminal degradation, e.g. Edman degradation}
- C12Q 1/37: involving peptidase or proteinase
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 21/76: Chemiluminescence; Bioluminescence
- G01N 21/648: {using evanescent coupling or surface plasmon coupling for the excitation of fluorescence}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2333/948: acting on peptide bonds (3.4)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention, in part, includes methods of single molecule protein sequencing that include using weak binding spectra in the amino acid identification.

## Claims

### Claim 1

A method of polypeptide sequencing comprising:
(a) contacting a polypeptide with a non-optimized plurality of low-affinity amino acid binders to allow binding of one or more binders of the non-optimized plurality to a N-terminal amino acid of the polypeptide, wherein the binding produces a specific detectable signal;
(b) identifying, based on the specific detectable signal, the N-terminal amino acid of the polypeptide, thereby generating an identified N-terminal amino acid; wherein identifying the N-terminal amino acid of the polypeptide comprises:
(i) kinetically measuring the specific detectable signal produced by the binding of the one or more binders to the N-terminal amino acid of the polypeptide, thereby generating kinetic measurements for the one or more binders;
(ii) combining the kinetic measurements;
(iii) determining a binding profile of the non-optimized plurality of low-affinity amino acid binders based at least in part on the combined kinetic measurements; and
(iv) identifying the N-terminal amino acid of the polypeptide from the binding profile of the non-optimized plurality of low-affinity amino acid binders;
(c) removing the identified N-terminal amino acid of the polypeptide thereby exposing a subsequent N-terminal amino acid of the polypeptide; and
(d) repeating steps (a) through (c) to identify the subsequent N-terminal amino acid of the polypeptide.

### Claim 2 (depends on 1)

The method of claim 1, further comprising repeating steps (c) and (d) a sufficient number of times to identify a portion or all of an amino acid sequence of the peptide.

### Claim 3 (depends on 1)

The method of claim 1, wherein the specific detectable signal is a fluorescent signal, a luminescent signal, or an electrical signal.

### Claim 4 (depends on 1)

The method of claim 1, wherein the detectable signal is detected using a single-photon avalanche diode (SPAD) detection method.

### Claim 5 (depends on 1)

The method of claim 1, further comprising determining specific detectable signals produced by binding of the one or more binders to the N-terminal amino acid and the subsequent N-terminal amino acid after removing the identified N-terminal amino acid of the polypeptide in (c).

### Claim 6 (depends on 5)

The method of claim 5, wherein determining the specific detectable signals comprises an optical detection method.

### Claim 7 (depends on 6)

The method of claim 6, wherein the optical detection method comprises microscopy.

### Claim 8 (depends on 5)

The method of claim 5, wherein determining the specific detectable signals comprises an electrical detection method.

### Claim 9 (depends on 8)

The method of claim 8, wherein the electrical detection method comprises a single-photon avalanche diode (SPAD) detection method.

### Claim 10 (depends on 5)

The method of claim 5, wherein determining the specific detectable signals produced by the binding of the one or more binders to the N-terminal amino acid occurs simultaneously.

### Claim 11 (depends on 5)

The method of claim 5, wherein determining the specific detectable signals produced by the binding of the one or more binders to the subsequent N-terminal amino acid occurs simultaneously.

### Claim 12 (depends on 1)

The method of claim 1, wherein the kinetically measuring comprises detecting a plurality of time-averaged specific detectable signals of the non-optimized plurality of low-affinity amino acid binders that bind the N-terminal amino acid of the polypeptide.

### Claim 13 (depends on 12)

The method of claim 12, wherein the detecting the plurality of time-averaged specific detectable signals comprises determining a length of time of the binding events of the non-optimized plurality of low-affinity amino acid binders that bind the N-terminal amino acid of the polypeptide.

### Claim 14 (depends on 1)

The method of claim 1, wherein the kinetically measuring comprises measuring using a high time resolution capable of detecting individual binding and unbinding events.

### Claim 15 (depends on 1)

The method of claim 1, wherein the kinetically measuring comprises measuring using a low time resolution capable of detecting and integrating signals of many binding and unbinding events to generate a plurality of detected time-averaged signals of the many binding and unbinding events, wherein a binding affinity is deduced based on the detected time-averaged signals of the many binding and unbinding events.

### Claim 16 (depends on 1)

The method of claim 1, wherein removing the identified N-terminal amino acid from the polypeptide comprises a cycle of Edman degradation.

### Claim 17 (depends on 1)

The method of claim 1, further comprising simultaneously detecting a plurality of produced specific detectable signals.

### Claim 18 (depends on 1)

The method of claim 1, wherein the non-optimized plurality of low-affinity amino acid binders comprises at least 5, 10, 15, 20 or more different binders.

### Claim 19 (depends on 1)

The method of claim 1, wherein each of the non-optimized plurality of low-affinity amino acid binders is a low-specificity binding reagent.