Research

Introduction

At the fundamental level; life is a collection of cells that house biochemical reactions; and biochemical reactions in turn are a collection of interacting biomolecules that undergo structural and chemical dynamics. Today, we do not possess the experimental means to visualize biochemical reactions at atomic resolution. This has limited our ability to build a truly predictive understanding regarding the elementary processes that govern living cells and ultimately give rise to disease. Thus, we do not fully understand what factors govern mutation probability during DNA replication in a test tube; nor are we able to predict mutational hotspots that give rise to diseases such as cancer. Similarly, we cannot predict from first principles whether or not a drug will bind to a given biomolecular target in a test tube or whether it will be an efficacious therapeutic in cells and in vivo. The goal of the Al-Hashimi laboratory is to develop new methods for ‘imaging’ the dynamics of nucleic acids at atomic resolution and to use this knowledge to help bridge the scales from the test tube, to cells, to organisms. Some of our overarching goals include quantitatively understanding the mechanisms that lead to genomic instability; how RNA folds into 3D structures at the atomic level; and to develop RNA and DNA targeting small molecule therapeutics to address diseases ranging from AIDS to cancer.

Development of methods for determining nucleic acid dynamic ensembles at atomic resolution: Role in molecular recognition and RNA folding

While it is now trivial to physically see cellular dynamics, such as a cell dividing, with a microscope, the technology does not yet exist to visualize the structural or chemical dynamics of biochemical reactions on an atomic level, even for the simplest molecules. We are developing hybrid experimental-computational methods that aim to accomplish this goal and determine dynamic-ensembles of nucleic acids at atomic resolution. Our effort thus far has led to the first 3D experimental visualization of motions in RNA at atomic resolution. We continue to improve the ensemble determination approaches as well as building frameworks for utilizing these dynamic ensemble descriptions to elucidate the mechanisms of RNA adaptive recognition; sequence-specific DNA-protein recognition; and RNA tertiary folding. Collaborators on these projects include Terry Oas (Duke); Dan Herschlag (Stanford); and Rick Russell (University of Texas-Austin). See this Movie of an unbound HIV-1 RNA dynamically sampling its many different drug-bound conformations.

Representative Publications:

  • Zhang Q Sun X Watt E W and Al-Hashimi HM (2006). Resolving the Motional Modes that Code for RNA Adaptation. Science 311: 653-6 PMID: 16456078
  • Zhang Q Stelzer AC Fisher CK and Al-Hashimi HM (2007). Visualizing Spatially Correlated Dynamics that Directs RNA Conformational Transitions. Nature 450:1263-7 PMID: 18097416
  • Bailor M Sun X and Al-Hashimi HM Topology Links RNA Secondary Structure with Global Conformation Dynamics and Adaptation Science 327(5962):202-6 2010

Fleeting conformational states of nucleic acids: Role in spontaneous mutations; RNA tertiary folding; and in the mechanisms of epitranscriptomics

In biology, some of the most functionally important conformational states of biomolecules exist in exceptionally low abundance (<0.01%); last for as little as a millionth of a second; and can involve subtle sub-angstrom movements of atoms. Our group has advanced approaches that combine NMR spectroscopic techniques with chemical traps for capturing such fleeting states in nucleic acids. This led to the discovery of rare nucleic acid conformational states that are implicated the mechanisms of spontaneous mutations; translational errors; viral replication; and RNA tertiary folding. We are examining the function of these fleeting states as well as their potential as opportune drug targets by using mutations to knock out or trap these states in in vitro and cell-based experiments. We are also investigating how epitranscriptomic modifications may exert their biological function by modulating the dynamics to these fleeting conformational states. Collaborators on these projects include Stacy Horner; Bryan Cullen; Chris Holly and Terry Oas at Duke. See this Movie for an example of fleeting base pair states that are thought to underlie spontaneous mutations.

Representative Publications:

  • Xue Y, Gracia B, Herschlag D, Russell R, and Al-Hashimi HM (2016) Visualizing Formation of an RNA Folding Intermediate through a Fast Highly Modular Secondary Structure Switch Nat Communications
  • Kimsey I, Petzold K, Sathyamoorthy B, Stein Z, and Al-Hashimi HM (2015). Visualizing Transient Watson-Crick Like Mispairs in DNA and RNA Duplexes. Nature 519(7543):315-20 PMID: 25762137
  • Dethoff L Petzold K Chugh J Casiano-Negroni A and Al-Hashimi HM (2012). Visualizing Transient Low-Populated Structures of RNA. Nature 491(7426):724-8 PMCID: PMC3590852

Hoogsteen Base Pairs: Redefining the DNA Double helix

Our group made the discovery that in canonical DNA duplexes, Watson-Crick base pairs exist in dynamic equilibrium with Hoogsteen base pairs in which the guanine or adenine base flips ~180 degrees to form a unique set of hydrogen bonds with its cytosine or thymine base complement. We are developing and applying methods based on solid state NMR and infrared (IR) spectroscopy to resolve Watson-Crick and Hoogsteen base pairs and their dynamic equilibria in nucleosomes and chromatin. We are examining the role of Hoogsteen base pairs in exposing the Watson-Crick face of nucleotide bases to mutagenic damage. We are also developing a new sequencing approach (‘Hoog-seq’) to map Hoogsteen base pairs genome-wide in vivo. We are interested in examining the enrichment of Hoogsteen base pairs relative to nucleosome positioning and other genomic features to assess potentially broader roles for Hoogsteen base pairs in gene expression and genome stability. Collaborators on these projects include the Jane and David Richardson, Kate Meyer, and David MacAlphine (Duke), Ioan Andricioaei (UC-Irvine), and Christopher Jaroniec (OSU). See this Movie to see the transitions between Watson-Crick and Hoogsteen base pairs.

Representative Publications:

  • Alvey HS Gottardo FL Nikolova EN Al-Hashimi HM Widespread Transient Hoogsteen Base-Pairs in Canonical Duplex DNA with Variable Energetics Nat Communications 5:4786 2014
  • Nikolova E Kim E Wise A O’Brien P Andricioaei I and Al-Hashimi HM (2011). Transient Hoogsteen Base-pairs in Canonical Duplex DNA. Nature 470(7335):498-502  PMCID: PMC3074620

RNA and DNA-targeted drug discovery

WWe are developing a dynamic-ensemble based virtual screening platform for enabling the rational discovery of nucleic acid targeting small molecule therapeutics. Our effort has so far focused on targeting functionally important non-coding RNA in HIV-1 as well as on the development of anti-cancer therapies targeting duplex DNA. By computationally docking a dynamic ensemble of RNA structures determined using our NMR-based methods, rather than a single static structure, we have overcome a major obstacle in structure-based RNA-targeted drug discovery. Docking to an HIV-1 RNA target ensemble resulted in the first computational discovery of an RNA-targeting small molecule with in vivo activity. We are also extending these approaches to target rare and unusual conformational states of nucleic acids that exist in low abundance (<1%) and for short periods of time (lifetimes on the order of milliseconds). These projects marry NMR spectroscopic, computational, in vitro and cell-based studies. Collaborators on these projects include Bryan Cullen and Amanda Hargrove (Duke). See this Movie for example of RNA ensemble used in computational docking.

Representative Publications:

  • Stelzer AC Frank AT Kratz JD Swanson M Gonzalez-Hernandez MJ Andricioaei I Markovitz DM and Al-Hashimi HM (2011). Discovery of HIV-1 Inhibitors by Targeting an RNA Dynamic Ensemble. Nat Chem Biol 7(8):553-9  PMCID: PMC3319144