# Exploring 3Dpol for RNA sequencing in real time

> **NIH NIH R21** · THOMAS JEFFERSON UNIVERSITY · 2021 · $195,000

## Abstract

Project Summary
 RNA sequencing (RNA-seq) in real time, known as the third-generation sequencing at the single-molecule
level, is an important technology that will improve our understanding of the human genome. The development of
real-time RNA-seq, however, has been challenging, due to the complexity of RNA in sequence and structure
that requires a processive reader with single-nucleotide resolution. While Pacific Biosciences (PacBio) can
generate long-reads, the process involves cDNA, which loses the informational content of RNA. The only real-
time RNA-seq in the current field that does not involved cDNA is the Oxford Nanopore Technology, which is
limited to sensing of 5-7 bases of RNA at a time. We report here enzymatic features of 3Dpol, the RNA-
dependent RNA polymerase of poliovirus, that are attractive for developing a new RNA-seq technology. We
show that 3Dpol copies the RNA template one base at a time with processivity across highly structured RNA.
We also show that 3Dpol prefers a hairpin primer to initiate RNA synthesis, generating a double-stranded (ds)-
hairpin RNA that allows sequencing of both the template strand and the complementary strand in a nanopore.
We further show that 3Dpol, when placed between two electrodes, displays protein conductance that is sensitive
to its conformational transition upon NTP-binding. We hypothesize that these features provide the basis to
explore 3Dpol for direct RNA-seq with single-nucleotide resolution at the single-molecule level. In Aim 1, we will
determine the ability and quality of 3Dpol as an enzymatic reader of RNA. We will test 3Dpol to read difficult RNA
sequences, including sequences that contain post-transcriptionally modified bases, homopolymers, and
repeated sequence motifs. We will determine the quality of RNA reading by 3Dpol using the Nanopore device in
a 2D (2-directional) platform that sequences both the template strand and the copied strand with the potential to
improve accuracy. These studies will also determine error signatures of 3Dpol that are useful for identification of
modified bases in RNA. In Aim 2, we consider that while the intrinsic error rate of 3Dpol is low (10-5), this quality
is masked in Nanopore sequencing, due to the latter’s technical error rate (10-15%). We will thus test the
possibility to develop 3Dpol in an electronic device for real-time sequencing of RNA by measuring protein
conductance through the polymerase. We will engineer 3Dpol to possess two built-in contacts for stable tethering
to two electrodes. We will measure protein conductance of 3Dpol in response to NTP binding using a scanning
tunneling microscope (STM). If successful, data of STM measurements will support a technology that will
generate long-reads of RNA-seq in a solid-state platform that produces direct electronic readout without the need
for dyes or labels. This work is at the forefront of exciting development of a new RNA-seq technology that will
broadly impact on RNA research and clini...

## Key facts

- **NIH application ID:** 10166895
- **Project number:** 5R21HG011120-02
- **Recipient organization:** THOMAS JEFFERSON UNIVERSITY
- **Principal Investigator:** Ya-Ming Hou
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $195,000
- **Award type:** 5
- **Project period:** 2020-05-18 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10166895

## Citation

> US National Institutes of Health, RePORTER application 10166895, Exploring 3Dpol for RNA sequencing in real time (5R21HG011120-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10166895. Licensed CC0.

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