# Facilitating the Chemistry of Fluorosequencing

> **NIH NIH R35** · UNIVERSITY OF TEXAS AT AUSTIN · 2024 · $384,421

## Abstract

ABSTRACT
While advances in nucleic acid sequencing to achieve parallel and single molecule analysis have been
astounding, analogous techniques for peptide/protein sequencing are lacking. Tandem mass-spec analysis still
requires millions of copies of a protein and is inherently serial rather than parallel. Thus, the current approaches
being applied to parallel and single molecule protein analysis primarily focus on the use of modified nanopores.
But their ability to differentiate subtle differences between individual amino acids (AAs), or strings of AAs, only
works in isolated cases. To create a parallel single molecule sequencing platform for peptides/proteins, in
collaboration with the Marcotte group, we devised a technique called fluorosequencing. In this technique, we
label AAs with fluorophores, and on a TIRF 4-channel microscope the N-terminal AAs are removed using classic
Edman degradation. As fluorophore labelled AAs are iteratively removed, both their identity and position are
revealed, generating a partial-sequence. The partial-sequences are compared to a genomic database of all
possible proteins, thereby revealing the proteins in the sample. Mixtures of millions of peptides are analyzed in
parallel; single molecules at a time. Albeit the method is functional, there are several aspects that require
improvement to generate a mature technology. One obstacle that we will overcome is the general lack of
approaches that allow sequential and selectively labelling of multiple amino acids on the same peptide/protein
with different tags, as well as differentiating the N-terminal and C-terminal residues from lysine and Glu/Asp AAs,
respectively. We will also explore labeling four AA within the following set: Cys, Lys, Tyr, Typ, His, Ser, Thr,
Glu/Asp, Arg, PSer, PThr, PTyr (P = phospho). Moreover, we feel that fluorosequencing can be readily extended
to post-translational modifications (PTMs) such as mono, di, and trimethylated Lys, as well as ubiquitination. The
sequential and selective labelling of four AAs and/or PTMs will involve conjugation handles carrying “click”
partners within a set of what we refer to as “click:clack” pairs, wherein the “clack” partner will carry one of a set
of four fluorophores appropriate for the four channels of our TIRF microscope. In some previous sequencing
runs we have discovered efficient donor/acceptor FRET between the fluorophores, thereby making the donor
intensity either weak or entirely invisible. To solve this problem, we will explore the use of designed fluorophores
whose emission can be turned on and off by intramolecular conjugate additions which are controlled by varying
the pH. In addition, because a series of common fluorophores, such as Cy and BODIPY dyes, do not survive the
TFA treatment used in Edman degradation, we are developing a base-induced method for N-terminal chain-end
sequencing. Importantly, while each thrust is focused upon use in fluorosequencing, the advances thereof are
broadly appli...

## Key facts

- **NIH application ID:** 10821396
- **Project number:** 5R35GM149308-02
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Eric V. Anslyn
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $384,421
- **Award type:** 5
- **Project period:** 2023-05-01 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10821396

## Citation

> US National Institutes of Health, RePORTER application 10821396, Facilitating the Chemistry of Fluorosequencing (5R35GM149308-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10821396. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*