# Single-molecule counting of circular RNAs using phage nanoparticles as surrogates

> **NIH NIH R21** · UNIVERSITY OF OKLAHOMA · 2020 · $189,469

## Abstract

Single-molecule counting of circular RNAs using phage nanoparticles as surrogates
Abstract
Circular RNA (circRNA) have been considered as promising breast cancer biomarkers, but the current qRT-PCR
and digital PCR techniques for quantifying them have some inherent limitations. T7 phage, a human-safe virus
nanoparticle specifically infecting bacteria, can be plated to infect bacteria in a plaque-forming assay to form
millimeter-scale plaques in a one to one format and at a single-particle resolution within 3 hours. Inspired from
this, we propose to develop a phage plaque counting (PPC) strategy that hires bioengineered fluorescent T7
phage as a surrogate to establish a one-to-one correspondence among target circRNA, phage nanoparticles,
and eye-visible phage-developed plaques, enabling us to simultaneously quantify multiple circRNA biomarkers
in a single test by simply counting the corresponding plaques. Briefly, a fluorescent phage probe with an
oligonucleotide (ONT-1) capable of capturing one unique segment of a target circRNA, and a magnetic
microparticle (MMP) probe with an ONT-2 capable of capturing another unique segment of the same target, co-
capture the target to form a sandwich complex where phages and target molecules are equimolar. Then the
phages are released and plated to develop fluorescent plaques in a one to one format, thus counting the plaques
at the single-particle level leads to visualized quantification of the target circRNA at a single-molecule level. A
fluorescent protein on the capsid makes the corresponding plaque fluoresce a unique color and enables the
simultaneous single-particle quantification of fluorescent plaques (in the same Petri dish) with each color coding
one target (i.e., by displaying green and red fluorescent protein for two corresponding targets). We hypothesize
that our PPC strategy with optimized conditions can simultaneously quantify a panel of two circRNAs as breast
cancer biomarkers in human serum with high sensitivity, specificity, and reproducibility. Aim 1: Establish and
optimize PPC strategy for quantifying single and multiple circRNA breast cancer biomarkers. We will
produce and purify the target circRNAs (circ_0001785 and circ_100219) by the overexpression method and use
the PPC method to quantify them with a series of dilutions in water. We will optimize the PPC and identify its
detection limit. Aim 2: Validate the PPC strategy for simultaneously quantifying multiple circRNA
biomarkers in breast cancer cells, tissues, and human serum. We will first use MMPs that can capture the
target circRNAs to magnetically remove the pre-existing target circRNAs from the commercial human serum,
which is then used to make serum samples with known concentrations of target circRNAs. Then we will employ
and optimize the PPC to quantify the target circRNAs in the serum. We will also isolate total RNAs from breast
cancer cells and in vitro breast tumor tissues to form aqueous RNA solutions by a commercial RNA isol...

## Key facts

- **NIH application ID:** 9877151
- **Project number:** 1R21EB028958-01
- **Recipient organization:** UNIVERSITY OF OKLAHOMA
- **Principal Investigator:** Binrui Cao
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $189,469
- **Award type:** 1
- **Project period:** 2020-02-01 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9877151

## Citation

> US National Institutes of Health, RePORTER application 9877151, Single-molecule counting of circular RNAs using phage nanoparticles as surrogates (1R21EB028958-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9877151. Licensed CC0.

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