R-loops are three-stranded nucleic acid structures that consist of an RNA-DNA hybrid and a displaced single-stranded DNA. They are found in a broad range of organisms, from bacteria to humans, and play roles in gene expression regulation, DNA and histone modification, generating antibody diversity, and DNA repair. Conversely, R-loops can also be a source of genomic instability, and they have been associated with various neurological diseases and cancers. Given this dichotomy, a clear understanding of R-loop formation and resolution is essential to comprehend how they play both healthy and harmful roles in cellular function. Although mounting evidence suggests that R-loop formation and resolution depend on torsion in the DNA molecule, the mechanics of this interaction are poorly understood due to a lack of appropriate investigation methodologies. A correlation between transcriptional pausing and R- loop formation has also been reported, however the transient nature of RNA-DNA hybrids complicate analysis of these dynamic interactions. Single-molecule studies are well-suited to investigate both R-loop formation and the effects of R-loops on transcription because they permit direct observation of dynamic processes. The goal of the proposed research is to elucidate R-loop dynamics and investigate their impact on transcription. This will be accomplished through two aims.
In Aim 1, the role of torsion in co-transcriptional R-loop formation will be identified. A combination of magnetic tweezers and single-molecule fluorescence techniques will be used to directly visualize the presence of RNA-DNA hybridization during transcription and investigate the relationship between negative DNA supercoiling and R-loop formation.
In Aim 2, the impact of R-loops on transcription will be characterized. R-loop hybridization will be directly visualized and the progression of RNA polymerase will be tracked, in real time, to investigate how R-loop formation may impact gene expression. This work will address fundamental gaps in knowledge concerning R-loop dynamics, and will help to clarify the roles that R-loops play in essential cellular processes such as transcription and gene expression. This essential information will help to define the molecular origin of a variety of human diseases that are connected with genetic instability, and will provide a foundation upon which to develop novel preventative and therapeutic treatments.

Public Health Relevance

R-loops are nucleic acid structures that can form during transcription, and although they are essential for some cellular processes, they are also associated with genetic instability and disease. The mechanisms of R-loop formation are poorly understood, and further research is required to better understand how they play both healthy and harmful roles in cellular function. Toward this goal, the proposed research focuses on the role of DNA topology, supercoiling in particular, in R-loop formation, and how the presence of R-loops impact transcription and gene expression.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Melillo, Amanda A
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Cornell University
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United States
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Baker, James E; Badman, Ryan P; Wang, Michelle D (2018) Nanophotonic trapping: precise manipulation and measurement of biomolecular arrays. Wiley Interdiscip Rev Nanomed Nanobiotechnol 10: