Consequences of Sequences: Kinetics and mechanics of nucleic acid interactions in sequence space
K. Sreenivasa (TU Delft - Applied Sciences)
C. Joo – Promotor (TU Delft - Applied Sciences)
S.M. Depken – Promotor (TU Delft - Applied Sciences)
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Abstract
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.
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