# Recognition of Synthetic Unnatural Base Pairs by RNA Polymerase

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2024 · $403,806

## Abstract

Project Summary/Abstract
 All life forms on Earth use the same set of natural genetic alphabets: adenine (A), cytosine (C), guanine
(G), thymine (T) (and uracil (U)) as the building blocks for storage and retrieval of their genetic information.
Recently, a major breakthrough was made in developing the first semi-synthetic organism that is able to store
and retrieve genetic information containing an unnatural base pair (UBP) in vivo. However, the molecular basis
of transcription processing of UBPs is poorly understood. An important and long-standing question remains
unanswered: How are these UBPs recognized by cellular transcription machinery? A lack of clear answers to
this important question represents a major knowledge gap in the field. The long-term goal of this project is to
tackle this important question. We hypothesize the transcription recognition of UBPs is governed by two layers
of specific interactions: specific interactions between the unnatural nucleic acid template and substrates as well
as their interplays with the active site of RNA polymerase. We will perform kinetic studies and compare the
transcription processing of three classes of representative UBPs by different RNA polymerases, including
single-subunit and multi-subunit RNA polymerases. We will determine the structural basis of transcription
recognition of UBPs and gain the mechanistic insights into how transcription machineries recognize unnatural
nucleotide substrate and catalyze the nucleotide addition reaction. We will utilize a combined approach that
includes X-ray crystallography, cryoEM, biophysics, biochemistry, computational biology, and nucleic acid
chemistry. The proposed research is significant and groundbreaking, because the novel knowledge and
structures obtained from this proposed research will have a transformative impact on the fields of transcription,
nucleic acid chemistry, as well as synthetic biology and vertically advance our understanding of the protein-
nucleic acid interactions and how unnatural nucleic acids and nucleotides are recognized by different RNA
polymerases. Ultimately, such knowledge will provide a framework for developing next generation of UBPs and
would produce novel therapeutic nucleic acids and proteins containing new functional groups.

## Key facts

- **NIH application ID:** 10809593
- **Project number:** 5R01GM148476-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Dong Wang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $403,806
- **Award type:** 5
- **Project period:** 2023-03-15 → 2027-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10809593

## Citation

> US National Institutes of Health, RePORTER application 10809593, Recognition of Synthetic Unnatural Base Pairs by RNA Polymerase (5R01GM148476-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10809593. Licensed CC0.

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