DNA/RNA interactions with a target are typically sequence-dependent: sequence informs structure, and structure in turn governs the interaction with a binding partner. Characterizing these interactions at high resolution and across large sequence spaces has so far been limited by the throughput of conventional single-molecule techniques. SPARXS, a recently developed method, addresses this by combining single-molecule fluorescence imaging with next-generation sequencing, enabling parallel measurement of sequence-dependent behavior across a library of DNA sequences.

In this thesis, I extend this approach to several new biological systems. I first show that the thrombin-binding aptamer exists in equilibrium between two distinct conformations, only one of which is competent for thrombin binding, pointing to a conformational selection mechanism that depends on buffer conditions and immobilization strategy. Building on this, I develop a single-molecule assay to characterize the binding kinetics of a library of RNA-based biotin aptamers towards biotin, uncovering sequence motifs beyond the reference sequence that retain binding affinity. I further develop a single-molecule assay to measure the kinetics of DNA looping across a library of looping sequences. Together, these assays establish SPARXS as a versatile platform for resolving how sequence shapes structure, dynamics, and recognition across diverse nucleic acid systems. ... Read more

Biological function is fundamentally determined by nucleic acid and protein sequence. Beyond encoding genetic information, nucleic acids also display complex physicochemical parameters that shape structure, dynamics, and interactions. Understanding how sequence variation sculpts the energetic landscapes underlying these properties requires methods that capture both molecular diversity and dynamic behavior. Single-molecule techniques are ideally suited to this task, but conventional formats remain time and cost intensive. Recent breakthroughs have enabled highly multiplexed approaches for observing molecular dynamics across millions of individual molecules representing thousands of sequences or barcoded entities. Though still in development, these methods have begun to bridge sequence, structure, dynamics, and function at scale, opening new opportunities in drug discovery, molecular diagnostics, and functional genomics. ... Read more