# High-throughput biochemistry with RNA-barcoded proteins

> **NIH NIH R21** · UNIVERSITY OF WASHINGTON · 2024 · $233,250

## Abstract

PROJECT SUMMARY
Vast numbers of mutations in human protein coding sequences have been identified by DNA
sequencing, but for only a tiny percentage of these variants do we understand the biochemical
basis for any defect. This project seeks to develop an approach to characterize the
biochemical activities – for such activities as thermostability, post-translational modification,
kinetics and catalysis – of protein variants at high throughput using the ease and scale of DNA
sequencing. Knowledge of biochemical activities can be used to annotate clinically-relevant
proteins, many of which have variants that are nearly all either unannotated or annotated as
variants of uncertain significance for their effect on pathogenicity. In the proposed approach,
each variant will be covalently linked in vivo to a unique RNA barcode by fusion to a tRNA
modifying enzyme that recognizes and couples to a short stem-loop RNA sequence. The
stem-loop will contain a barcode sequence that identifies the variant. The barcodes allow a
pool of variants to be subjected to a biochemical assay, with the results read out by DNA
sequencing of the barcodes. By developing this technology to express variant proteins in vivo,
we allow these proteins to fold or become post-translationally modified in their native cellular
environment; to bind to other cellular proteins or ligands; to become modified upon cellular
perturbation; or to be synthesized in a variety of hosts, including human cultured cells. As
proof-of-concept examples of the use of this method, in Aim 1, variants of dihydrofolate
reductase will be assessed for thermal stability. The variants will be fused to the tRNA
modifying enzyme and expressed in E. coli; the fusion proteins will be purified in a pooled
format; and aliquots of the proteins will be heated to varying temperatures followed by
purification of the soluble, undenatured protein (thermal proteome profiling). The number of
sequence reads of the soluble fraction of each variant allows melting temperatures to be
determined. In Aim 2, we will develop this method in mammalian cells to quantify the
abundance of protein variants of thiopurine methyltransferase. In summary, we will develop a
high throughput in vivo protein barcoding method to study fundamental biochemical properties
of variant proteins that have so far remained inaccessible by current methods.

## Key facts

- **NIH application ID:** 10856629
- **Project number:** 1R21HG013504-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** STANLEY FIELDS
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $233,250
- **Award type:** 1
- **Project period:** 2024-07-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10856629

## Citation

> US National Institutes of Health, RePORTER application 10856629, High-throughput biochemistry with RNA-barcoded proteins (1R21HG013504-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10856629. Licensed CC0.

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