CAREER: A Novel Framework for Measuring and Engineering Twisting and Writhing in DNA

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Genes are the basic units of biological code inside of every living cell. The genome of every cell has thousands of genes, arranged in distinct, spatial patterns that give rise to complex, coordinated genetic programs. Each time a gene is turned on, the twisted, double helical structure of DNA must be uncoiled, to expose single-stranded DNA for RNA synthesis (transcription). As thousands of genes are simultaneously transcribed, the displaced twists of the DNA travel across the genome, forming a living, fluid landscape of DNA writhing. This project will develop new biotechnology to reveal how the dynamics of persistent twisting in DNA alters cellular activity and cell fate. The overarching goal is to understand the functional relationship between different spatial arrangements of genetic programs and the fluid landscape of DNA twisting. Using a combination of techniques from synthetic biology, experimental biophysics, and control theory, the work will allow researchers to experimentally visualize and model localized DNA twisting, in concert with gene transcription. The results are expected to provide practical understanding of how to spatially design synthetic neighborhoods of genes and ultimately, synthetic genomes that could be used in biotechnology. Research opportunities will be provided for graduate and undergraduate students. In addition, in collaboration teachers in the Santa Ynez, Santa Maria and Lompoc school districts in California, the project will develop curricula, hands-on training workshops, educational videos, and interactive life-sized spatial models to promote understanding of the programmability and the dynamics of DNA. Biologists have previously studied the migratory dynamics of twisting in relatively short strands of DNA. This project aims to simultaneously visualize and integrate models for the processes of DNA twisting and gene transcription, which historically have been studied separately on different timescales. The PI has recently functionalized and integrated a magnetic tweezer fluorescence microscopy system with cell-free reactors in small-volume capillary tubes. This system, combined with an array of recently developed genetic circuits and reporter constructs, will be used to study of effects of DNA writhing and twisting on transcription. Simultaneously, the formation of higher-order DNA knots in linear and circular DNA, such as plectonemes, will be visualized. These are new in vitro methods for measuring how local supercoiling density alters or controls nearby gene transcription. The project also will develop new in vivo biosensors that measure the degree of localized twist and writhe in circular DNA in living cells, to link in vivo supercoiling to nearby transcriptional activity. In addition, biophysical models will be developed to describe how the spatial distribution, transport, and insulation of localized supercoiling controls transcription in DNA.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.