Publications
133 results found
Cardo L, Karunatilaka KS, Rueda D, et al., 2012, Single molecule FRET characterization of large ribozyme folding., Methods Mol Biol, Vol: 848, Pages: 227-251
A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg(2+) and Ca(2+)) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments.
Aleman EA, Rueda D, 2011, 2-Aminopurine Single-Molecule Fluorescence, 55th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 474-474, ISSN: 0006-3495
Wood S, Kulshina N, Ferre-D'Amare A, et al., 2011, Single Molecule Studies of the c-di-GMP Riboswitch, 55th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 177-177, ISSN: 0006-3495
Lamichhane R, Baker KA, Cunningham PR, et al., 2011, Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly, 55th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 233-233, ISSN: 0006-3495
Karunatilaka KS, Solem A, Pyle AM, et al., 2010, Single-molecule analysis of Mss116-mediated group II intron folding, NATURE, Vol: 467, Pages: 935-U75, ISSN: 0028-0836
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- Citations: 63
Lamichhane R, Solem A, Black W, et al., 2010, Single-molecule FRET of protein-nucleic acid and protein-protein complexes: Surface passivation and immobilization, METHODS, Vol: 52, Pages: 192-200, ISSN: 1046-2023
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- Citations: 75
Zhao R, Marshall M, Aleman EA, et al., 2010, Laser-Assisted Single-Molecule Refolding (LASR), BIOPHYSICAL JOURNAL, Vol: 99, Pages: 1925-1931, ISSN: 0006-3495
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- Citations: 19
Hsieh J, Koutmou KS, Rueda D, et al., 2010, A divalent cation stabilizes the active conformation of the B. subtilis RNase P x pre-tRNA complex: a role for an inner-sphere metal ion in RNase P., J Mol Biol, Vol: 400, Pages: 38-51
Metal ions interact with RNA to enhance folding, stabilize structure, and, in some cases, facilitate catalysis. Assigning functional roles to specifically bound metal ions presents a major challenge in analyzing the catalytic mechanisms of ribozymes. Bacillus subtilis ribonuclease P (RNase P), composed of a catalytically active RNA subunit (PRNA) and a small protein subunit (P protein), catalyzes the 5'-end maturation of precursor tRNAs (pre-tRNAs). Inner-sphere coordination of divalent metal ions to PRNA is essential for catalytic activity but not for the formation of the RNase P x pre-tRNA (enzyme-substrate, ES) complex. Previous studies have demonstrated that this ES complex undergoes an essential conformational change (to the ES* conformer) before the cleavage step. Here, we show that the ES* conformer is stabilized by a high-affinity divalent cation capable of inner-sphere coordination, such as Ca(II) or Mg(II). Additionally, a second, lower-affinity Mg(II) activates cleavage catalyzed by RNase P. Structural changes that occur upon binding Ca(II) to the ES complex were determined by time-resolved Förster resonance energy transfer measurements of the distances between donor-acceptor fluorophores introduced at specific locations on the P protein and pre-tRNA 5' leader. These data demonstrate that the 5' leader of pre-tRNA moves 4 to 6 A closer to the PRNA x P protein interface during the ES-to-ES* transition and suggest that the metal-dependent conformational change reorganizes the bound substrate in the active site to form a catalytically competent ES* complex.
Lamichhane R, Daubner GM, Thomas-Crusells J, et al., 2010, RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 4105-4110, ISSN: 0027-8424
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- Citations: 80
Karunatilaka KS, Solem A, Pyle A-M, et al., 2010, Single-Molecule Analysis of Mss116-Mediated Group II Intron Folding, Publisher: CELL PRESS, Pages: 472A-472A, ISSN: 0006-3495
Christian T, Romano L, Rueda D, 2010, Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution, Publisher: CELL PRESS, Pages: 437A-438A, ISSN: 0006-3495
Aleman EA, Pedini HS, Rueda D, 2010, Covalent-Bond-Based Immobilization Approaches for Single-Molecule Fluorescence, Publisher: CELL PRESS, Pages: 185A-185A, ISSN: 0006-3495
Lamichhane R, Daubner GM, Thomas-Crusells J, et al., 2010, RNA Looping By PTB: Evidence Using Fret and NMR Spectroscopy and For a Role in Splicing Repression, Publisher: CELL PRESS, Pages: 72A-73A, ISSN: 0006-3495
Christian TD, Romano LJ, Rueda D, 2009, Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 106, Pages: 21109-21114, ISSN: 0027-8424
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- Citations: 73
Aleman EA, Pedini HS, Rueda D, 2009, Covalent-Bond-Based Immobilization Approaches for Single-Molecule Fluorescence, CHEMBIOCHEM, Vol: 10, Pages: 2862-2866, ISSN: 1439-4227
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- Citations: 15
Guo Z, Karunatilaka KS, Rueda D, 2009, Single-molecule analysis of protein-free U2-U6 snRNAs, Nature Structural and Molecular Biology, Vol: 16, Pages: 1154-1159, ISSN: 1545-9985
Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2–U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2–U6 from yeast and mutants of highly conserved regions of U2–U6. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent, and we provide evidence suggesting that the second step corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that have an important role in splicing.
Zhao R, Rueda D, 2009, RNA folding dynamics by single-molecule fluorescence resonance energy transfer, METHODS, Vol: 49, Pages: 112-117, ISSN: 1046-2023
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- Citations: 73
Karunatilaka KS, Rueda D, 2009, Single-molecule fluorescence studies of RNA: A decade's progress, CHEMICAL PHYSICS LETTERS, Vol: 476, Pages: 1-10, ISSN: 0009-2614
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- Citations: 14
Rueda D, Lamichhane R, Auweter SD, et al., 2009, Evidence of RNA looping by PTB using Fluorescence Resonance Energy Transfer and NMR spectroscopy, Publisher: FEDERATION AMER SOC EXP BIOL, ISSN: 0892-6638
Steiner M, Rueda D, Sigel RKO, 2009, Ca2+ induces the formation of two distinct subpopulations of group II intron molecules., Angew Chem Int Ed Engl, Vol: 48, Pages: 9739-9742
The folding pathway of the Sc.ai5γ derived group II intron ribozyme D135 is highly specific to the correct M2+ cofactor. Upon partial replacement of Mg2+ with Ca2+, the molecules split into two distinct static subpopulations that are not interchangeable. Type 2 molecules thereby form a compact but misfolded state.
Coller J, Rueda D, 2009, RNA research in the rustbelt., RNA Biol, Vol: 6, Pages: 9-11
Deep in the heart of Ohio, scientists from across the Midwest gathered in October to share their latest findings and highlight the strength of RNA research in the heartland. Represented were researchers from Delaware, Indiana, Kentucky, Michigan, Ohio, Pennsylvania and West Virginia. With over 220 participants, the 2008 annual Rustbelt RNA Meeting (RRM) was the largest gathering of this group in its 10-year history. The success of this year's RRM lies on the extraordinary efforts of organizers Dawn Chandler (Ohio State University) and Girish Shukla (Cleveland State University).
Aleman EA, Lamichhane R, Rueda D, 2008, Exploring RNA folding one molecule at a time, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 12, Pages: 647-654, ISSN: 1367-5931
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- Citations: 46
Ditzler MA, Rueda D, Mo J, et al., 2008, A rugged free energy landscape separates multiple functional RNA folds throughout denaturation., Nucleic Acids Res, Vol: 36, Pages: 7088-7099
The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.
Steiner M, Karunatilaka KS, Sigel RKO, et al., 2008, Single-molecule studies of group II intron ribozymes (Proceedings of the National Academy of Sciences of the United States of America (2008) 105, 37, (13853-13858) DOI: 10.1073/pnas.0804034105), Proceedings of the National Academy of Sciences of the United States of America, Vol: 105, ISSN: 0027-8424
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- Citations: 3
Zhang X, Wigley DB, 2008, The 'glutamate switch' provides a link between ATPase activity and ligand binding in AAA plus proteins, Nature Structural and Molecular Biology, Vol: 15, Pages: 1223-1227, ISSN: 1545-9985
AAA+ proteins carry out diverse functions in cells. In most cases, their ATPase activity is tightly regulated by protein partners and target ligands, but the mechanism for this control has remained unclear. We have identified a conserved link between the ligand binding and ATPase sites in AAA+ proteins. This link, which we call the 'glutamate switch', regulates ATPase activity directly in response to the binding of target ligands by controlling the orientation of the conserved glutamate residue in the DExx motif, switching it between active and inactive conformations. The reasons for this level of control of the ATPase activity are discussed in the context of the biological processes catalyzed by AAA+ proteins.
Steiner M, Karunatilaka KS, Sigel RKO, et al., 2008, Single-molecule studies of group II intron ribozymes, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 105, Pages: 13853-13858, ISSN: 0027-8424
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- Citations: 82
Ditzler MA, Alemán EA, Rueda D, et al., 2007, Focus on function: single molecule RNA enzymology., Biopolymers, Vol: 87, Pages: 302-316, ISSN: 0006-3525
The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.
Bobeck MJ, Rueda D, Walter NG, et al., 2007, Structural modeling of sequence specificity by an autoantibody against single-stranded DNA., Biochemistry, Vol: 46, Pages: 6753-6765, ISSN: 0006-2960
11F8 is a sequence-specific pathogenic anti-single-stranded (ss)DNA autoantibody isolated from a lupus prone mouse. Site-directed mutagenesis of 11F8 has shown that six binding site residues (R31VH, W33VH, L97VH, R98VH, Y100VH, and Y32VL) contribute 80% of the free energy for complex formation. Mutagenesis results along with intermolecular distances obtained from fluorescence resonance energy transfer were implemented here as restraints to model docking between 11F8 and the sequence-specific ssDNA. The model of the complex suggests that aromatic stacking and two sets of bidentate hydrogen bonds between binding site arginine residues (R31VH and R96VH) and loop nucleotides provide the molecular basis for high affinity and specificity. In part, 11F8 utilizes the same ssDNA binding motif of Y32VL, H91VL, and an aromatic residue in the third complementarity-determining region to recognize thymine-rich sequences as do two anti-ssDNA autoantibodies crystallized in complex with thymine. R31SVH is a dominant somatic mutation found in the J558 germline sequence that is implicated in 11F8 sequence specificity. A model of the mutant R31S11F8.ssDNA complex suggests that different interface contacts occur when serine replaces arginine 31 at the binding site. The modeled contacts between the R31S11F8 mutant and thymine are closely related to those observed in other anti-ssDNA binding antibodies, while we find additional contacts between 11F8 and ssDNA that involve amino acids not utilized by the other antibodies. These data-driven 11F8.ssDNA models provide testable hypotheses concerning interactions that mediate sequence specificity in 11F8 and the effects of somatic mutation on ssDNA recognition.
Gondert ME, Tinsley RA, Rueda D, et al., 2006, Catalytic core structure of the trans-acting HDV ribozyme is subtly influenced by sequence variation outside the core., Biochemistry, Vol: 45, Pages: 7563-7573, ISSN: 0006-2960
The human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication. Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformation with a specific AP76 stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence, suggesting that AP76 becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of 3' product concentration yet Mg2+ dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of the 5' sequence and Mg2+ ions.
Rueda D, Walter NG, 2006, Fluorescent energy transfer readout of an aptazyme-based biosensor., Methods Mol Biol, Vol: 335, Pages: 289-310, ISSN: 1064-3745
Biosensors are devices that amplify signals generated from the specific interaction between a receptor and an analyte of interest. RNA structural motifs called aptamers have recently been discovered as receptor components for biosensors owing to the ease with which they can be evolved in vitro to bind a variety of ligands with high specificity and affinity. By coupling an aptamer as allosteric control element to a catalytic RNA such as the hammerhead ribozyme, ligand binding is transduced into a catalytic event. We have made use of fluorescence resonance energy transfer (FRET) to further amplify ligand induced catalysis into an easily detectable fluorescence signal. This chapter reviews in detail the methods and protocols to prepare a theophylline specific aptazyme and to label its substrate with fluorophores. We also include detailed protocols to characterize by FRET the binding affinity of the target, theophylline, as well as the external substrate to the aptazyme. The chapter should therefore facilitate the implementation of RNA-based biosensor components for other analytes of interest.
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