Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding

Abstract

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Singlemolecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg 2+ ). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1−10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.

Selective Detection and Characterization of Small Cysteine-Containing Peptides with Cluster-Modified Nanopore Sensing

“…Further discussion of this phenomenon can be found elsewhere. 67 To minimize effects from peptide aggregation at these higher concentrations, peptide solutions were typically discarded after 1 week.…”

“…The peptide concentration at the pore is greatly reduced due to diffusion effects, and we estimate it to be on the order of 10 μM. Further discussion of this phenomenon can be found elsewhere . To minimize effects from peptide aggregation at these higher concentrations, peptide solutions were typically discarded after 1 week.…”

Atomistic Picture of Opening–Closing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations

“…An intriguing structural aspect of HJs in solution is their conformational transitions between the stacked (closed) and open states depending on ionic strength. − The transitions enable sequence-specific junction cleavage by resolvases and permit branch migration in the open state. − ,,, Basic characteristics of HJ opening–closing transitions were obtained from the studies of free HJs under different ionic environments. These studies revealed that the open state is prevalent at low monovalent cation concentration (<40 mM), while the closed state becomes preferred at higher monovalent concentrations (>150 mM) and in the presence of divalent cations. ,,, Although the factors governing the balance between open and closed HJs have been elucidated, ,,, the atomistic details of the opening–closing process remain elusive due to the fast dynamics of the process and the resolution limitations. ,, The commonly assumed two-state model for opening and closing ignores the structural complexity and conformational flexibility of HJs. For example, there are two possible stacking patterns among the HJ’s arms which comprise the closed conformation, resulting in two isomers (I and II) which can interexchange via the open state (Figure ).…”

DNA Holliday Junction: History, Regulation and Bioactivity

“…As a transient DNA intermediate, HJ performs its biological function mainly relying on conformational variation [ 12 ]. Generally, HJ generally has two configurations: “X-stacked” and “open-planar” ( Figure 1 B) [ 13 , 14 , 15 ]. In the presence of low salt concentrations, the “open-planar” state is a predominant configuration; otherwise, the junction can be transformed to a symmetric X-shape termed “X-stacked” at higher ionic concentrations.…”