Abstract/Summary
Despite the nearly 4 decades of technology development for biomolecular structure analysis since the discovery
of the ribozyme, researchers still grapple with an inability to characterize important intermediates and other
aspects of functional nucleic acid structure. Although powerful approaches such as X-ray crystallography, NMR,
and Cryo-EM can provide atomic-level detail, they are essentially blind to structural transformations, co-existing
solution structures and conformational flexibility. Time-resolved NMR provides useful information about large
conformation changes on long timescales; however, it is not able to detail the conformational ensemble along
a folding pathway. Mass spectrometry (MS) including MS coupled with ion mobility spectrometry (IMS) are
promising tools to provide additional structural detail but suffer from two disadvantages for studying functional
nucleic acids. The first is a relatively low signal intensity associated with the analysis of oligonucleotides in
negative ion mode. This potentially renders many important structures (e.g., low population) essentially invisible
to the analysis. The second is that the ions can undergo rapid structural transformation such as compaction in
the gas phase making the extraction of solution-relevant details very difficult. Overall, MS analysis requires
dramatically increased sensitivity and better means to extract solution structure information to characterize
important intermediates and conformational flexibility in structure establishment. Here we propose the
development of a paradigm-breaking, front-end technology platform for enhanced compound ionization and
improved structure resolution. The technology is built upon the new ionization technique capillary Vibrating
Sharp-edge Spray Ionization (cVSSI), which is shown to provide 10-to-100-fold ion signal enhancements over
state-of-the-art ESI sources in negative ion mode. This work will extend that advantage by another order of
magnitude to break through the sensitivity issues associated with the characterization of functional nucleic
acids. Additionally, the work will produce an integrated platform that utilizes new, rapid mixing strategies for
performing hydrogen-deuterium exchange labeling kinetics as well as oligonucleotide folding kinetics. The
microfluidics device will couple seamlessly with cVSSI to accomplish high sensitivity ionization as well as to
permit pulsed (in-droplet and on-line) and continuous (on-line) HDX labeling. With the device it will immediately
be possible to resolve co-existing structures, structural transformations, and conformer flexibility based on
differences in HDX reactivity. A novel HDX kinetics modeling methodology will also be developed to allow for
structure elucidation from even the shortest timescale measurements. The new technology platform will be
validated with gold standard functional nucleic acids including 10-23 DNAzyme, Hairpin Ribozyme, and Group
II Intron systems. The complete mapping of conformational heterogeneity and flexibilities and folding processes
of these systems will open the floodgates for further work on therapeutically-important functional nucleic acids.
