Probing RNA structure and dynamics using nanopore and next generation sequencing.

It has become increasingly evident that the structures RNAs adopt are conformationally dynamic; the various structured states that RNAs sample govern their interactions with other nucleic acids, proteins, and ligands to regulate a myriad of biological processes. Although several biophysical approaches have been developed and used to study the dynamic landscape of structured RNAs, technical limitations have limited their application to all classes of RNA due to variable size and flexibility. Recent advances combining chemical probing experiments with next-generation- and direct sequencing have emerged as an alternative approach to exploring the conformational dynamics of RNA. In this review, we provide a methodological overview of the sequencing-based techniques used to study RNA conformational dynamics. We discuss how different techniques have enabled us to better understand the propensity of RNAs from a variety of different classes to sample multiple conformational states. Finally, we present examples of the ways these techniques have reshaped how we think about RNA structure.

Nuclease resistance and protein recognition properties of DNA and hybrid PNA-DNA four-way junctions.

Nucleic acids possess unique biochemical features that make them ideal candidates to inhibit "difficult to target" proteins. The limited stability of nucleic acids in vivo presents a major obstacle to their development as drugs. Here, immobile four-way junctions (4WJs) are used to target the DNA-binding cytokine, High Mobility Group B1. Hybrid 4WJs composed of DNA and peptide nucleic acids (PNA) are investigated. PNA possess enhanced nuclease stability vs. DNA. 4WJs are incubated with Exonuclease III and DNase I. The nuclease assays show that 4WJs containing multiple PNAs possess significantly higher stability. Circular dichroism assays are used to probe the groove topology of 4WJs with the minor groove binder, DAPI. The CD data indicates that multi-PNA 4WJs possess altered minor groove dimensions that reduces DAPI binding affinity. Logic suggests that the minor groove of multi-PNA hybrids possess significant perturbations to the topology and local electrostatic environment that prevents proper binding/recognition by nucleases and thus enhances stability.