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Principal Investigator: Peng Li
Organization: WEST VIRGINIA UNIVERSITY
Fiscal Year: 2024
Award: $526,400
Funding agency: National Institute of General Medical Sciences
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.
Terms: <3-D structure><3-dimensional structure><3D structure><Acoustics><Address><Blood capillaries><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 era><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 period><COVID-19 public health crisis><COVID-19 years><Catalytic DNA><Catalytic RNA><Cell Communication and Signaling><Cell Signaling><Characteristics><Complex><Coupled><Coupling><Cryo-electron Microscopy><Cryoelectron Microscopy><DNAzymes><Data><Data Analyses><Data Analysis><Data Set><Deoxyribozymes><Deuterium><Deuterium Exchange Measurement><Development><Devices><Diagnostic><Disadvantaged><Disease><Disorder><Electron Cryomicroscopy><Ensure><FRET><Fluorescence Resonance Energy Transfer><Förster Resonance Energy Transfer><Gases><H element><H2 isotope><Heterogeneity><Hydrogen><Hydrogen-Deuterium Exchange Measurement><Intervening Sequences><Intracellular Communication and Signaling><Introns><Investigators><Ions><Kinetics><Label><Ligand Binding><Maps><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measurement><Methodology><Methods><Microfluidic Device><Microfluidic Lab-On-A-Chip><Microfluidic Microchips><Modeling><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><Nucleic Acids><Oligo><Oligonucleotides><Pathway interactions><Performance><Phase><Physiologic pulse><Population><Procedures><Process><Pulse><RNA Stability><Research><Research Personnel><Researchers><Resolution><Ribozymes><Roentgen Rays><Role><Running><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Signal Transduction><Signal Transduction Systems><Signaling><Single Crystal Diffraction><Solvents><Source><Specificity><Spectrometry><Stream><Structure><System><Systems Integration><Techniques><Technology><Therapeutic><Time><Visualization><Work><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><biological signal transduction><blind><capillary><computerized data processing><conformation><conformational><conformational state><conformationally><conformations><conformer><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><cryo-EM><cryoEM><cryogenic electron microscopy><data exchange><data interpretation><data processing><data transfer><data transmission><design><designing><developmental><diagnostic tool><flexibility><flexible><hairpin ribozyme><human disease><improved><insight><instrumentation><integrated system><interest><ion mobility><ionization><ionization method><ionization technique><kinetic model><microfluidic chip><new diagnostics><new drug treatments><new drugs><new pharmacological therapeutic><new technology><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><new treatment approach><new treatment strategy><next generation diagnostics><next generation therapeutics><novel><novel diagnostics><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel technologies><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutics><novel therapy><novel therapy approach><nucleic acid structure><oligos><pathway><post-COVID><post-COVID-19><post-coronavirus disease 2019><process optimization><resolutions><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><social role><structural biology><system integration><tech development><technology development><technology platform><technology system><three dimensional structure><tool><vibration>