DNA is both a fundamental building block of life and a versatile natural polymer. A myriad of DNA-binding proteins carry out their biological function by recognizing specific shapes and structures of DNA, or by actively altering the DNA configuration through force generation. Single-molecule techniques are ideally suited for studying these interactions owing to their ability to follow dynamic processes in real time, while precisely exerting and measuring force on the system of interest.

The packaging of genomic DNA into nucleosomes is a ubiquitous feature of eukaryotic genomes. Conventionally, nucleosomes have been viewed as DNA storage units that obstruct the accessibility of DNA to the transcription machinery, thereby suppressing gene expression. However, the nucleosome’s unique topology and its hierarchical organization into chromatin predict a multifaceted regulatory repertoire encoded by its physical traits.

Genomic DNA is a crowded track shared by many motor proteins and decorated with a plethora of roadblocks. Proper traffic management is critical to the maintenance of genome integrity and control of gene activity. However, the molecular rules and functional consequences of motor collisions and roadblock encounters remain poorly understood. The lab has built an experimental pipeline—leveraging biochemical reconstitution and single-molecule visualization—to interrogate these dynamic events in real time.

In parallel to developing in vitro single-molecule assays to visualize genomic machines in action, we also sought to understand their behavioral patterns in vivo on a genome-wide scale. Our group developed a novel transcriptomic method termed SEnd-seq (simultaneous 5'- and 3'-end RNA sequencing) to study gene expression in bacteria. Through an optimized and unbiased protocol that involves a key single-strand circularization step, SEnd-seq captures the sequence of both termini of the same RNA molecule and enables the profiling of full-length transcripts.

We have applied expertise in single-molecule methodology to many other biological problems. Leveraging the power of single-molecule techniques in capturing transient, stochastic, and heterogeneous molecular events, these studies provide mechanistic insights into dynamic biological processes that are obscured in bulk assays due to ensemble averaging.

The discovery of the double-helical structure of DNA ushered in a new era of molecular biology. Among the many experimental approaches to study protein-DNA interactions, single-molecule techniques are frequently chosen for their unique capability of following transient and heterogeneous molecular events in an asynchronous population, which allows the user to delineate the time-dependent behavior of individual molecules or complexes in exquisite detail.