# A Functional genomics platform with integrated library cloning and molecular display

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2021 · $350,603

## Abstract

DNA sequencing technologies have transformed our ability to define genomes and transcriptomes.
Techniques to study the proteins they encode, and thus our ability to define new protein functions, discover
biologic medicines, and determine disease mechanisms, however, have not similarly advanced. This focused
technology development project will provide new methodologies to enable inexpensive, high throughput
functional studies of expressed protein libraries.
Aim 1: Develop methodology for LASSO-based ORFeome cloning from RNA
We propose to adapt our previously established Long Adapter Single Stranded Oligonucleotide (“LASSO”)
probe-based DNA sequence capture platform to the cloning of expressed ORFeomes directly from tissue RNA.
A reverse transcriptase-mediated gap filling reaction will be defined and combined with an RNA-templated DNA-
DNA ligation reaction, to enable LASSO cloning of ORF sequences while maintaining their correct translational
reading frame. Captured ORFeome libraries may be shuttled into any expression vector of choice, including in
vitro display vectors, like that developed in Aim 2. As validation, a complete human transcriptomic LASSO probe
library will be designed, constructed, tested and characterized, prior to being shared with the research
community.
Aim 2. Develop a self-assembling, barcoded-RNA display format for full length proteins
In vitro display technologies permit affinity-based screening of complex protein libraries. High throughput DNA
sequencing, when applied to library screening, provides a powerful means to quantify binding interactions. We
have previously developed a system that combines DNA sequencing with ribosome display for the ParalleL
Analysis of Translated ORFeomes (“PLATO”). The ribosome display format imposes significant limitations,
however, which we propose to overcome by developing a novel self-assembling barcoded-RNA version of
PLATO (“sabr-PLATO”). Rigorous validation experiments will be used to extensively characterize the
performance of the sabr-PLATO protein screening platform.
 Rapid production of ORFeome libraries using LASSO cloning from tissue-derived RNA, combined with robust
screening of the encoded protein libraries using sabr-PLATO, will provide a means for any laboratory to
functionally mine the proteome of any tissue from any organism. The synergistic, yet independent Aims of this
project utilize the interdisciplinary expertise of the co-PIs, and build upon previous studies successfully carried
out in their laboratories.

## Key facts

- **NIH application ID:** 10226125
- **Project number:** 5R01GM127353-04
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Harry Benjamin Larman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $350,603
- **Award type:** 5
- **Project period:** 2018-08-15 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10226125

## Citation

> US National Institutes of Health, RePORTER application 10226125, A Functional genomics platform with integrated library cloning and molecular display (5R01GM127353-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10226125. Licensed CC0.

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