# Macromolecular Crowding effects on DNA mechanics, topology and transcription

> **NIH NIH R35** · EMORY UNIVERSITY · 2023 · $384,431

## Abstract

Effects of macromolecular crowding on DNA mechanics, topology, transcription, and condensation
ABSTRACT
Macromolecular crowding (MMC) changes the concentration and affinities of intracellular biomolecules and
promotes liquid phase separation. MMC has been shown to change the melting temperature of DNA oligos, but
broad characterization of how it affects the mechanical stability of DNA is incomplete. Crowded DNA
condensates may generate sub-piconewton retractile tension on DNA, which can be conveniently explored using
magnetic/optical tweezers. While many experiments on DNA motors employ tensions opposing or assisting
translocation of several to tens of pN, our group showed that sub-piconewton tension affects DNA topology, from
supercoiling to protein-mediated looping, as well as the probability that an elongating E. coli RNA polymerase
(RNAP) surpasses a protein roadblock. Surprisingly, the effect of MMC on topologies such as supercoiling and
protein-mediated loops, and processes such as transcription, protein spreading, and condensation has not been
well characterized. This proposal aims to assess the effects of MMC on DNA configurations including unwinding
and looping, protein spreading, and liquid phase separation to integrate these features into our understanding
of intracellular molecular biology. To do so, we integrate single-molecule, in vitro experiments with in vivo
measurements and computational/theoretical approaches
Over the next five years, we will analyze both model and/or novel systems with single-molecule techniques to
learn how MMC changes DNA structure, affects protein-mediated looping, and alters transcription. We will also
investigate how MMC influences ParB-mediated spreading along DNA and liquid-liquid phase separation (LLPS)
which requires crowding agents in vitro. Then we propose to build artificial LLPS systems with which to learn
what components are required to localize a liquid-liquid phase separated droplet on a DNA segment. P-granules,
Cajal bodies, segrosome, and the nucleolus are some examples of LLPS that include specific genomic regions
and demonstrate the ubiquity and importance of this phenomenon. Macromolecular crowding generates forces
that affect fundamental DNA mechanics and topology and in the last decade MMC has emerged as a driver of
LLPS. We will integrate in vitro experiments with computational and theoretical approaches and compare with
appropriate in vivo measurements performed by a collaborator. Discovering the mechanisms by which crowding
modifies DNA configurations, transactions, and segregation will advance our understanding of genome
biophysics and regulation and provide new tools for synthetic biology.

## Key facts

- **NIH application ID:** 10623720
- **Project number:** 1R35GM149296-01
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** Laura Finzi
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $384,431
- **Award type:** 1
- **Project period:** 2023-05-01 → 2028-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10623720

## Citation

> US National Institutes of Health, RePORTER application 10623720, Macromolecular Crowding effects on DNA mechanics, topology and transcription (1R35GM149296-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10623720. Licensed CC0.

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