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Frank A.T.,University of California at Irvine | Frank A.T.,Nymirum Inc. | Horowitz S.,University of Michigan | Andricioaei I.,University of California at Irvine | And 2 more authors.
Journal of Physical Chemistry B | Year: 2013

The development of methods for predicting NMR chemical shifts with high accuracy and speed is increasingly allowing use of these abundant, readily accessible measurements in determining the structure and dynamics of proteins. For nucleic acids, however, despite the availability of semiempirical methods for predicting 1H chemical shifts, their use in determining the structure and dynamics has not yet been examined. Here, we show that 1H chemical shifts offer powerful restraints for RNA structure determination, allowing discrimination of native structure from non-native states to within 2-4 Å, and <3 Å when highly flexible residues are ignored. Theoretical simulations shows that although 1H chemical shifts can provide valuable information for constructing RNA dynamic ensembles, large uncertainties in the chemical shift predictions and inherent degeneracies lead to higher uncertainties as compared to residual dipolar couplings. © 2013 American Chemical Society. Source


Stelzer A.C.,University of Michigan | Stelzer A.C.,Nymirum Inc. | Frank A.T.,University of California at Irvine | Kratz J.D.,University of Michigan | And 6 more authors.
Nature Chemical Biology | Year: 2011

Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (Ki values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC50 ∼1/423.1 μM). © 2011 Nature America, Inc. All rights reserved. Source


Xue Y.,Duke University | Kellogg D.,Duke University | Kimsey I.J.,Duke University | Sathyamoorthy B.,Duke University | And 4 more authors.
Methods in Enzymology | Year: 2015

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states. © 2015 Elsevier Inc. All rights reserved. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 215.02K | Year: 2014

DESCRIPTION (provided by applicant): The central dogma of molecular biology has undergone a significant revision where RNAs have emerged as important regulators of cell activity. Astonishingly, as much as 80% of human DNA is transcribed into RNA as recently reported by the Encyclopedia of DNA Elements (ENCODE) consortium. New classes of RNA are being identified including many which are non-protein coding (ncRNA). Nonetheless ncRNAs are involved in regulating cellular activity. Deciphering the structure-function relationship of these RNAs is of immediate importance as ncRNAs have now been implicated in a variety of diseases including myotonic dystrophy type 1, prostate cancer, spinal muscular atrophy, and Huntington's disease-like 2. Traditional approaches todetermine high-resolution RNA 3D structures have proven to be prohibitively time consuming and expensive. Nymirum has developed an Automated RNA 3D Structure Determination System that is faster, cheaper, and more robust than current approaches. This


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.46M | Year: 2015

DESCRIPTION provided by applicant The central dogma of molecular biology has undergone a significant revision where RNAs have emerged as important regulators of cell activity Astonishingly as much as of human DNA is transcribed into RNA as recently reported by the Encyclopedia of DNA Elements ENCODE consortium New classes of RNA are being identified including many which are non protein coding ncRNA Nonetheless ncRNAs are involved in regulating cellular activity Deciphering the structure function relationship of these RNAs is of immediate importance as ncRNAs have now been implicated in a variety of diseases including myotonic dystrophy type prostate cancer spinal muscular atrophy and Huntingtonandapos s disease like Traditional approaches to determine high resolution RNA D structures have proven to be prohibitively time consuming and expensive Nymirum has developed an Automated RNA D Structure Determination System that is faster cheaper and more robust than current approaches This System will be commercialized for use by the drug industry and academic researchers in their quest to understand and treat human disease PUBLIC HEALTH RELEVANCE RNA D structure and function information is critical to understanding basic cell function delineating dysfunctional RNA contribution to human disease and finding new drugs to treat diseases caused by dysfunctional RNAs Nymirum has developed an Automated RNA D Structure Determination System that is faster cheaper and more robust than current approaches

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