# An integrated toolkit for real-time analysis of coupled nascent transcription

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2024 · $424,583

## Abstract

Project Summary
 Proper regulation of gene expression is required for cellular function. Conversely, dysregulation of gene
expression is a major cause of disease. Furthermore, expression of two genes is frequently coupled. Coupled
expression is expected if two genes respond to the same signal or are expressed from a bidirectional promoter
(BDP). About one in ten human genes are controlled by a BDP, defined as two genes with less than 1 kilobase
between their transcription start sites. However, coupled expression in general and BDPs in particular remain
very poorly understood.
 The premise of this proposal is that our current lack of understanding of coupled expression is due to a
lack of tools. The goal of this proposal therefore is to develop an integrated experimental and computational
toolkit for real-time analysis of coupled nascent transcription. For example, we currently do not understand if
transcription of two BDP-controlled genes is synergistic, antagonistic, or independent in quantity and in time.
More generally, understanding coupled expression in both quantity and time requires real-time single-molecule
imaging of nascent transcription integrated with computational modeling.
 Here we propose to develop an experimental platform that allows direct visualization of nascent RNA of
two genes (Aim 1). Specifically, we will adapt RNA hairpin systems from bacteriophage to allow real-time
visualization and counting of single nascent RNAs in live cells. We will evaluate multiple microscope modalities
and optimize a microscopy platform for long-term gentle live-cell imaging at high spatiotemporal resolution.
Next, we will develop computational methods for quantification of nascent BDP transcription imaging data (Aim
2). We will also develop Bayesian Inference methods for inference of two-gene coupled nascent transcription
data, and identify the minimal mathematical models required to understand BDP transcription. Next, we will
apply these new experimental and computational tools for an initial exploration of BDPs (Aim 3). Specifically,
we will take a two-pronged approach exploring multiple BDPs at a safe-harbor locus as well as dedicated
studies of the endogenous NIPBL BDP. This will serve as proof-of-concept for our integrated toolkit.
 In summary, we propose to establish an integrated experimental and computational toolkit for real-time
analysis of coupled nascent transcription. We will devote significant efforts to disseminate the experimental
and computational tools and anticipate this being useful beyond BDP studies. We anticipate this toolkit will
allow future mechanistic studies to better understand BDPs, will make it possible to establish BDPs as
powerful tools for synthetic biology, and empower studies of coupled nascent transcription beyond BDPs.

## Key facts

- **NIH application ID:** 10778870
- **Project number:** 1R01EB035127-01
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Anders Sejr Hansen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $424,583
- **Award type:** 1
- **Project period:** 2024-06-01 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10778870

## Citation

> US National Institutes of Health, RePORTER application 10778870, An integrated toolkit for real-time analysis of coupled nascent transcription (1R01EB035127-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10778870. Licensed CC0.

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