Investigating RNA Hairpin Stability and Protein Binding Using Optical Tweezers

Abstract

More than 1.2 million people in the US are living with HIV. This RNA virus must be reverse‐transcribed into DNA before it can be integrated into the host genome and subsequently replicated. Retroviral DNA synthesis is generally understood, but questions remain about many identified critical elements. The transactivation response (TAR) RNA hairpin, specifically, inhibits the minus‐strand transfer step in reverse transcription and thereby regulates the life cycle of the HIV‐1 retrovirus. Before proceeding with reverse transcription, the TAR hairpin must be destabilized: chaperone assistance from the nucleocapsid (NC) protein can stimulate this rate‐limiting step 3000‐fold. Recent interest in the folding landscape of the TAR hairpin has revealed in‐vitro thermodynamic properties in the presence and absence of NC. These discoveries were enabled by optical tweezers force spectroscopy, the nature of which requires a complex TAR hairpin construct. Unfortunately, design and synthesis of the necessary TAR hairpin construct has limited the quantity and diversity of thermodynamic analyses regarding this essential facet of HIV‐1. We aim to develop efficient protocols to produce TAR RNA hairpin constructs optimized for optical tweezer manipulations.In order to manipulate individual hairpin molecules, the optical tweezers require that the RNA hairpin be ligated to two long double‐stranded DNA “handles.” One handle of the final construct specifically binds a bead in the optical tweezer dual‐laser trap, while the other handle binds a second bead that is manipulated via a micropipette. Tiny displacements of the second bead in the laser trap are used to calculate the force required to unzip the hairpin, and the thermodynamic parameters for force‐induced unfolding. The two ~3,000 base pair handles were constructed by amplification of bacterial plasmid DNA by PCR, restricted to create specific sticky ends, and purified by gel electrophoresis. RNA hairpins were transcribed from short DNA templates that were themselves synthesized by automated solid‐phase synthesis with specific sequences for RNA pol binding and the hairpin structure of interest. The handles were then individually ligated via several short DNA and RNA linker sequences to the RNA hairpin. Gel purification between ligation steps removed alternative ligation products, allowing size‐based verification and isolation of intermediates and the complete hairpin construct. Initial unzipping data support the correct length and composition of the final construct. We anticipate that subsequent data will contribute to a broader understanding of RNA hairpin stability as well as provide useful insight about a potential HIV‐1 drug target.Support or Funding InformationNIH‐NIGMS (Grant 2R01‐GM072462‐10), NSF‐MCB (Grant 1213883), Camille and Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.