# Large-Scale, Quantitative Protein Affinity Assays on a High-Throughput DNA Sequencing Chip

> **NIH NIH R43** · PROTILLION BIOSCIENCES, INC. · 2020 · $247,800

## Abstract

Abstract
The high-throughput DNA sequencing revolution has brought a match between the scale of nucleic acid
quantification and the complexity of human biology. However, despite expectation that similarly-widespread
and scalable methods for probing protein function would emerge, most protein functional assays and
mutational analyses remain largely low to medium throughput. While multiplexed protein methods exist, they
tend to be employed only by a small number of “specialists” because 1) they have not reached the immense
scalability of DNA sequencing methods, and 2) they are technically challenging to implement. Protillion is
commercializing a platform for high-throughput protein analysis built directly on the ubiquitous Illumina
sequencer, demonstrating a path to bringing protein functional characterization into the high-throughput era.
This protein mapping platform (Prot-MaP) enables the generation of tens of millions of clusters of immobilized
proteins directly on DNA sequencing flow cell through efficient tethered in-situ transcription and translation.
Fluorescence-based protein functional assays can then be performed directly on this protein array, with results
quantified by highly sensitive fluorescence imaging. Prot-MaP enables the generation of immense mutational
datasets for both peptides and full-length functional proteins, allowing high-throughput analysis of mutational
effects based on direct biophysical observations of protein function. Unlike directed evolution methods this
platform allows for direct, quantitative measurements of the function of entire protein libraries, and a shorter
time-to-result in a single experiment on automated, widely distributed instrumentation. We will deploy the Prot-
MaP platform in two different modes – one using small, ~15 amino acid peptides as affinity reagents, and
another displaying larger, ~115 amino acid nanobodies, to characterize the biophysical interactions between T
Cell immunoreceptor with Ig and ITIM domains, or TIGIT protein. TIGIT is an ideal example target, as it is
highly soluble, commercially available, and has diagnostic (for detecting HIV progression) and therapeutic (in
combination with PD1 blockade in cancer immunotherapy) application potential. We plan to analyze our high-
throughput data sets 1) to understand the benefits afforded pre-selection of peptides compared to rational
engineering of peptide libraries, 2) to identify clades of peptides and nanobodies that form different families of
binders, 3) to test the identification of these distinct families that bind distinct epitopes on TIGIT experimentally,
and 4) assess the salt and temperature dependence of binding, 5) examine the dependence of specific key
peptides to binding using structural information. The identification of nanobodies and peptides that bind distinct
regions of TIGIT will open the door to creation of both sandwich-style affinity assays, as well as high affinity
ligands (via avidity) for potential therapeutic ...

## Key facts

- **NIH application ID:** 10007027
- **Project number:** 1R43GM137655-01
- **Recipient organization:** PROTILLION BIOSCIENCES, INC.
- **Principal Investigator:** Curtis Layton
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $247,800
- **Award type:** 1
- **Project period:** 2020-05-01 → 2020-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10007027

## Citation

> US National Institutes of Health, RePORTER application 10007027, Large-Scale, Quantitative Protein Affinity Assays on a High-Throughput DNA Sequencing Chip (1R43GM137655-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10007027. Licensed CC0.

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