Co-reporter:David E. Condon, Scott D. Kennedy, Brendan C. Mort, Ryszard Kierzek, Ilyas Yildirim, and Douglas H. Turner
Journal of Chemical Theory and Computation 2015 Volume 11(Issue 6) pp:2729-2742
Publication Date(Web):April 16, 2015
DOI:10.1021/ct501025q
Molecular dynamics (MD) simulations for RNA tetramers r(AAAA), r(CAAU), r(GACC), and r(UUUU) are benchmarked against 1H–1H NOESY distances and 3J scalar couplings to test effects of RNA torsion parametrizations. Four different starting structures were used for r(AAAA), r(CAAU), and r(GACC), while five starting structures were used for r(UUUU). On the basis of X-ray structures, criteria are reported for quantifying stacking. The force fields, AMBER ff99, parmbsc0, parm99χ_Yil, ff10, and parmTor, all predict experimentally unobserved stacks and intercalations, e.g., base 1 stacked between bases 3 and 4, and incorrect χ, ϵ, and sugar pucker populations. The intercalated structures are particularly stable, often lasting several microseconds. Parmbsc0, parm99χ_Yil, and ff10 give similar agreement with NMR, but the best agreement is only 46%. Experimentally unobserved intercalations typically are associated with reduced solvent accessible surface area along with amino and hydroxyl hydrogen bonds to phosphate nonbridging oxygens. Results from an extensive set of MD simulations suggest that recent force field parametrizations improve predictions, but further improvements are necessary to provide reasonable agreement with NMR. In particular, intramolecular stacking and hydrogen bonding interactions may not be well balanced with the TIP3P water model. NMR data and the scoring method presented here provide rigorous benchmarks for future changes in force fields and MD methods.
Co-reporter:Salvatore F. Priore, Andrew D. Kauffmann, Jayson R. Baman, and Douglas H. Turner
Biochemistry 2015 Volume 54(Issue 22) pp:3413-3415
Publication Date(Web):May 21, 2015
DOI:10.1021/bi501564d
Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments encode more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg2+-dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that interactions of the ribosome with the pseudoknot may affect the level of translation for PB1-F2 and N40.
Co-reporter:Jonathan L. Chen, Stanislav Bellaousov, Jason D. Tubbs, Scott D. Kennedy, Michael J. Lopez, David H. Mathews, and Douglas H. Turner
Biochemistry 2015 Volume 54(Issue 45) pp:6769-6782
Publication Date(Web):October 9, 2015
DOI:10.1021/acs.biochem.5b00833
Knowledge of RNA structure is necessary to determine structure–function relationships and to facilitate design of potential therapeutics. RNA secondary structure prediction can be improved by applying constraints from nuclear magnetic resonance (NMR) experiments to a dynamic programming algorithm. Imino proton walks from NOESY spectra reveal double-stranded regions. Chemical shifts of protons in GH1, UH3, and UH5 of GU pairs, UH3, UH5, and AH2 of AU pairs, and GH1 of GC pairs were analyzed to identify constraints for the 5′ to 3′ directionality of base pairs in helices. The 5′ to 3′ directionality constraints were incorporated into an NMR-assisted prediction of secondary structure (NAPSS-CS) program. When it was tested on 18 structures, including nine pseudoknots, the sensitivity and positive predictive value were improved relative to those of three unrestrained programs. The prediction accuracy for the pseudoknots improved the most. The program also facilitates assignment of chemical shifts to individual nucleotides, a necessary step for determining three-dimensional structure.
Co-reporter:Jonathan L. Chen, Scott D. Kennedy, and Douglas H. Turner
Biochemistry 2015 Volume 54(Issue 21) pp:3269-3285
Publication Date(Web):April 24, 2015
DOI:10.1021/acs.biochem.5b00012
Influenza A is an RNA virus with a genome of eight negative sense segments. Segment 7 mRNA contains a 3′ splice site for alternative splicing to encode the essential M2 protein. On the basis of sequence alignment and chemical mapping experiments, the secondary structure surrounding the 3′ splice site has an internal loop, adenine bulge, and hairpin loop when it is in the hairpin conformation that exposes the 3′ splice site. We report structural features of a three-dimensional model of the hairpin derived from nuclear magnetic resonance spectra and simulated annealing with restrained molecular dynamics. Additional insight was provided by modeling based on 1H chemical shifts. The internal loop containing the 3′ splice site has a dynamic guanosine and a stable imino (cis Watson–Crick/Watson–Crick) GA pair. The adenine bulge also appears to be dynamic with the A either stacked in the stem or forming a base triple with a Watson–Crick GC pair. The hairpin loop is a GAAA tetraloop closed by an AC pair.
Co-reporter:Tian Jiang, Scott D. Kennedy, Walter N. Moss, Elzbieta Kierzek, and Douglas H. Turner
Biochemistry 2014 Volume 53(Issue 32) pp:5236-5248
Publication Date(Web):July 15, 2014
DOI:10.1021/bi500611j
Influenza A virus utilizes RNA throughout infection. Little is known, however, about the roles of RNA structures. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure starts 79 nucleotides downstream of the M2 mRNA 5′ splice site. Here, a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multibranch structure of this RNA. Imino proton NMR spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ∼14 °C higher than that for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.
Co-reporter:David E. Condon, Ilyas Yildirim, Scott D. Kennedy, Brendan C. Mort, Ryszard Kierzek, and Douglas H. Turner
The Journal of Physical Chemistry B 2014 Volume 118(Issue 5) pp:1216-1228
Publication Date(Web):December 30, 2013
DOI:10.1021/jp408909t
Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced.
Co-reporter:Douglas H. Turner
Biopolymers 2013 Volume 99( Issue 12) pp:1097-1104
Publication Date(Web):
DOI:10.1002/bip.22294
ABSTRACT
Interpreting the tsunami of sequence information for RNA would be facilitated by an understanding of all the physical principles determining RNA structure. In principle, a complete understanding would make it computationally possible to find RNA sequences that fold for function and to predict their three-dimensional structure. It would, thus, also facilitate discovery of new principles relating structure to function. This review covers some of the progress in understanding RNA over roughly the preceding 40 years and suggests progress still to be made. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 1097–1104, 2013.
Co-reporter:Ilyas Yildirim, Scott D. Kennedy, Harry A. Stern, James M. Hart, Ryszard Kierzek, and Douglas H. Turner
Journal of Chemical Theory and Computation 2012 Volume 8(Issue 1) pp:172-181
Publication Date(Web):December 1, 2011
DOI:10.1021/ct200557r
All-atom force fields are important for predicting thermodynamic, structural, and dynamic properties of RNA. In this paper, results are reported for thermodynamic integration calculations of free energy differences of duplex formation when CG pairs in the RNA duplexes r(CCGG)2, r(GGCC)2, r(GCGC)2, and r(CGCG)2 are replaced by isocytidine–isoguanosine (iCiG) pairs. Agreement with experiment was improved when ε/ζ, α/γ, β, and χ torsional parameters in the AMBER99 force field were revised on the basis of quantum mechanical calculations. The revised force field, AMBER99TOR, brings free energy difference predictions to within 1.3, 1.4, 2.3, and 2.6 kcal/mol at 300 K, respectively, compared to experimental results for the thermodynamic cycles of CCGG → iCiCiGiG, GGCC → iGiGiCiC, GCGC → iGiCiGiC, and CGCG → iCiGiCiG. In contrast, unmodified AMBER99 predictions for GGCC → iGiGiCiC and GCGC → iGiCiGiC differ from experiment by 11.7 and 12.6 kcal/mol, respectively. In order to test the dynamic stability of the above duplexes with AMBER99TOR, four individual 50 ns molecular dynamics (MD) simulations in explicit solvent were run. All except r(CCGG)2 retained A-form conformation for ≥82% of the time. This is consistent with NMR spectra of r(iGiGiCiC)2, which reveal an A-form conformation. In MD simulations, r(CCGG)2 retained A-form conformation 52% of the time, suggesting that its terminal base pairs may fray. The results indicate that revised backbone parameters improve predictions of RNA properties and that comparisons to measured sequence dependent thermodynamics provide useful benchmarks for testing force fields and computational methods.
Co-reporter:Jonathan L. Chen, Abigael L. Dishler, Scott D. Kennedy, Ilyas Yildirim, Biao Liu, Douglas H. Turner, and Martin J. Serra
Biochemistry 2012 Volume 51(Issue 16) pp:3508-3522
Publication Date(Web):April 10, 2012
DOI:10.1021/bi3002709
Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5′GG/3′UU motif were added to the single representation in the previous database. This revises the ΔG°37 for the 5′GG/3′UU motif from an unfavorable 0.5 kcal/mol to a favorable −0.2 kcal/mol. Similarly, the ΔG°37 for the 5′UG/3′GU motif changes from 0.3 to −0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°37, ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.
Co-reporter:Scott D. Kennedy, Ryszard Kierzek, and Douglas H. Turner
Biochemistry 2012 Volume 51(Issue 46) pp:
Publication Date(Web):November 7, 2012
DOI:10.1021/bi301372t
The RNA duplex, (5′GACGAGUGUCA)2, has two conformations in equilibrium. The nuclear magnetic resonance solution structure reveals that the major conformation of the loop, 5′GAGU/3′UGAG, is novel and contains two unusual Watson–Crick/Hoogsteen GG pairs with G residues in the syn conformation, two A residues stacked on each other in the center of the helix with inverted sugars, and two bulged-out U residues. The structure provides a benchmark for testing approaches for predicting local RNA structure and a sequence that allows the design of a unique arrangement of functional groups and/or a conformational switch into nucleic acids.
Co-reporter:
Biochemistry 2011 Volume 50(Issue 5) pp:640-653
Publication Date(Web):December 6, 2010
DOI:10.1021/bi101470n
Three-way multibranch loops (junctions) are common in RNA secondary structures. Computer algorithms such as RNAstructure and MFOLD do not consider the identity of unpaired nucleotides in multibranch loops when predicting secondary structure. There is limited experimental data, however, to parametrize this aspect of these algorithms. In this study, UV optical melting and a fluorescence competition assay are used to measure stabilities of multibranch loops containing up to five unpaired adenosines or uridines or a loop E motif. These results provide a test of our understanding of the factors affecting multibranch loop stability and provide revised parameters for predicting stability. The results should help to improve predictions of RNA secondary structure.
Co-reporter:Ilyas Yildirim, Harry A. Stern, Jason D. Tubbs, Scott D. Kennedy, and Douglas H. Turner
The Journal of Physical Chemistry B 2011 Volume 115(Issue 29) pp:9261-9270
Publication Date(Web):July 1, 2011
DOI:10.1021/jp2016006
Accurately modeling unpaired regions of RNA is important for predicting structure, dynamics, and thermodynamics of folded RNA. Comparisons between NMR data and molecular dynamics simulations provide a test of force fields used for modeling. Here, NMR spectroscopy, including NOESY, 1H–31P HETCOR, DQF-COSY, and TOCSY, was used to determine conformational preferences for single-stranded GACC RNA. The spectra are consistent with a conformational ensemble containing major and minor A-form-like structures. In a series of 50 ns molecular dynamics (MD) simulations with the AMBER99 force field in explicit solvent, initial A-form-like structures rapidly evolve to disordered conformations. A set of 50 ns simulations with revised χ torsions (AMBER99χ force field) gives two primary conformations, consistent with the NMR spectra. A single 1.9 μs MD simulation with the AMBER99χ force field showed that the major and minor conformations are retained for almost 68% of the time in the first 700 ns, with multiple transformations from A-form to non-A-form conformations. For the rest of the simulation, random-coil structures and a stable non-A-form conformation inconsistent with NMR spectra were seen. Evidently, the AMBER99χ force field improves structural predictions for single-stranded GACC RNA compared to the AMBER99 force field, but further force field improvements are needed.
Co-reporter:Ilyas Yildirim, Harry A. Stern, Scott D. Kennedy, Jason D. Tubbs and Douglas H. Turner
Journal of Chemical Theory and Computation 2010 Volume 6(Issue 5) pp:1520-1531
Publication Date(Web):April 16, 2010
DOI:10.1021/ct900604a
A reparameterization of the torsional parameters for the glycosidic dihedral angle, χ, for the AMBER99 force field in RNA nucleosides is used to provide a modified force field, AMBER99χ. Molecular dynamics simulations of cytidine, uridine, adenosine, and guanosine in aqueous solution using the AMBER99 and AMBER99χ force fields are compared with NMR results. For each nucleoside and force field, 10 individual molecular dynamics simulations of 30 ns each were run. For cytidine with AMBER99χ force field, each molecular dynamics simulation time was extended to 120 ns for convergence purposes. Nuclear magnetic resonance (NMR) spectroscopy, including one-dimensional (1D) 1H, steady-state 1D 1H nuclear Overhauser effect (NOE), and transient 1D 1H NOE, was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine. The AMBER99 force field overestimates the population of syn conformations of the base orientation and of C2′-endo sugar puckering of the pyrimidines, while the AMBER99χ force field’s predictions are more consistent with NMR results. Moreover, the AMBER99 force field prefers high anti conformations with glycosidic dihedral angles around 310° for the base orientation of purines. The AMBER99χ force field prefers anti conformations around 185°, which is more consistent with the quantum mechanical calculations and known 3D structures of folded ribonucleic acids (RNAs). Evidently, the AMBER99χ force field predicts the structural characteristics of ribonucleosides better than the AMBER99 force field and should improve structural and thermodynamic predictions of RNA structures.
Co-reporter:Biao Liu, Neelaabh Shankar and Douglas H. Turner
Biochemistry 2010 Volume 49(Issue 3) pp:
Publication Date(Web):November 18, 2009
DOI:10.1021/bi901541j
RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 °C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1−stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson−Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures.
Co-reporter:Nicholas B. Hammond, Blanton S. Tolbert, Ryszard Kierzek, Douglas H. Turner and Scott D. Kennedy
Biochemistry 2010 Volume 49(Issue 27) pp:
Publication Date(Web):May 20, 2010
DOI:10.1021/bi100332r
Thermodynamic stabilities of 2 × 2 nucleotide tandem AG internal loops in RNA range from −1.3 to +3.4 kcal/mol at 37 °C and are not predicted well with a hydrogen-bonding model. To provide structural information to facilitate development of more sophisticated models for the sequence dependence of stability, we report the NMR solution structures of five RNA duplexes: (rGACGAGCGUCA)2, (rGACUAGAGUCA)2, (rGACAAGUGUCA)2, (rGGUAGGCCA)2, and (rGACGAGUGUCA)2. The structures of these duplexes are compared to that of the previously solved (rGGCAGGCC)2 (Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449−4460). For loops bounded by Watson−Crick pairs, the AG and Watson−Crick pairs are all head-to-head imino-paired (cis Watson−Crick/Watson−Crick). The structures suggest that the sequence-dependent stability may reflect non-hydrogen-bonding interactions. Of the two loops bounded by G-U pairs, only the 5′UAGG/3′GGAU loop adopts canonical UG wobble pairing (cis Watson−Crick/Watson−Crick), with AG pairs that are only weakly imino-paired. Strikingly, the 5′GAGU/3′UGAG loop has two distinct duplex conformations, the major of which has both guanosine residues (G4 and G6 in (rGACGAGUGUCA)2) in a syn glycosidic bond conformation and forming a sheared GG pair (G4-G6*, GG trans Watson−Crick/Hoogsteen), both uracils (U7 and U7*) flipped out of the helix, and an AA pair (A5-A5*) in a dynamic or stacked conformation. These structures provide benchmarks for computational investigations into interactions responsible for the unexpected differences in loop free energies and structure.
Co-reporter:Ruiting Liang, Elzbieta Kierzek, Ryszard Kierzek, and Douglas H. Turner
Biochemistry 2010 Volume 49(Issue 37) pp:
Publication Date(Web):June 17, 2010
DOI:10.1021/bi100286n
Microarrays with isoenergetic pentamer and hexamer 2′-O-methyl oligonucleotide probes with LNA (locked nucleic acid) and 2,6-diaminopurine substitutions were used to probe the binding sites on the RNase P RNA specificity domain of Bacillus subtilis. Unexpected binding patterns were revealed. Because of their enhanced binding free energies, isoenergetic probes can break short duplexes, merge adjacent loops, and/or induce refolding. This suggests new approaches to the rational design of short oligonucleotide therapeutics but limits the utility of microarrays for providing constraints for RNA structure determination. The microarray results are compared to results from chemical mapping experiments, which do provide constraints. Results from both types of experiments indicate that the RNase P RNA folds similarly in 1 M Na+ and 10 mM Mg2+.
Co-reporter:Ilyas Yildirim, Harry A. Stern, Jiri Sponer, Nada Spackova and Douglas H. Turner
Journal of Chemical Theory and Computation 2009 Volume 5(Issue 8) pp:2088-2100
Publication Date(Web):July 2, 2009
DOI:10.1021/ct800540c
Guanine-adenine (GA) base pairs play important roles in determining the structure, dynamics, and stability of RNA. In RNA internal loops, GA base pairs often occur in tandem arrangements and their structure is context and sequence dependent. Calculations reported here test the thermodynamic integration (TI) approach with the amber99 force field by comparing computational predictions of free energy differences with the free energy differences expected on the basis of NMR determined structures of the RNA motifs (5′-GCGGACGC-3′)2, (5′-GCiGGAiCGC-3′)2, (5′-GGCGAGCC-3′)2, and (5′-GGiCGAiGCC-3′)2. Here, iG and iC denote isoguanosine and isocytidine, which have amino and carbonyl groups transposed relative to guanosine and cytidine. The NMR structures show that the GA base pairs adopt either imino (cis Watson−Crick/Watson−Crick A-G) or sheared (trans Hoogsteen/Sugar edge A-G) conformations depending on the identity and orientation of the adjacent base pair. A new mixing function for the TI method is developed that allows alchemical transitions in which atoms can disappear in both the initial and final states. Unrestrained calculations gave ΔG° values 2−4 kcal/mol different from expectations based on NMR data. Restraining the structures with hydrogen bond restraints did not improve the predictions. Agreement with NMR data was improved by 0.7 to 1.5 kcal/mol, however, when structures were restrained with weak positional restraints to sample around the experimentally determined NMR structures. The amber99 force field was modified to partially include pyramidalization effects of the unpaired amino group of guanosine in imino GA base pairs. This provided little or no improvement in comparisons with experiment. The marginal improvement is observed when the structure has potential cross-strand out-of-plane hydrogen bonding with the G amino group. The calculations using positional restraints and a nonplanar amino group reproduce the signs of ΔG° from the experimental results and are, thus, capable of providing useful qualitative insights complementing the NMR experiments. Decomposition of the terms in the calculations reveals that the dominant terms are from electrostatic and interstrand interactions other than hydrogen bonds in the base pairs. The results suggest that a better description of the backbone is key to reproducing the experimental free energy results with computational free energy predictions.
Co-reporter:Elzbieta Kierzek, Anna Pasternak, Karol Pasternak, Zofia Gdaniec, Ilyas Yildirim, Douglas H. Turner and Ryszard Kierzek
Biochemistry 2009 Volume 48(Issue 20) pp:
Publication Date(Web):April 6, 2009
DOI:10.1021/bi9002056
Locked nucleic acids (LNA) considerably enhance the thermodynamic stability of DNA and RNA duplexes. We report the thermodynamic stabilities of LNA-2′-O-methyl RNA/RNA duplexes designed to provide insight into the contributions of stacking and hydrogen bonding interactions to the enhanced stability. The results show that hydrogen bonding of LNA nucleotides is similar to that of 2′-O-methyl RNA nucleotides, whereas the 3′-stacking interactions are on average ∼0.7 kcal/mol more favorable at 37 °C than for 2′-O-methyl or RNA nucleotides. Moreover, NMR spectra suggest helical preorganization of the single-stranded tetramer, CLAMALUM, probably due to restriction of some torsion angles. Thus, enhanced stacking interactions and helical preorganization of single-stranded oligonucleotides contribute to the extraordinary stabilization of duplexes by LNA nucleotides.
Co-reporter:Gang Chen, Scott D. Kennedy and Douglas H. Turner
Biochemistry 2009 Volume 48(Issue 24) pp:
Publication Date(Web):June 1, 2009
DOI:10.1021/bi8019405
RNA internal loops are often important sites for folding and function. Residues in internal loops can have pKa values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the and nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 × 3 nucleotide and larger size-symmetric internal loops. These and nearest neighbors, with CA adjacent to a closing Watson−Crick pair, are further stabilized when the pH is lowered from 7 to 5.5. The results are consistent with a significantly larger fraction (from ∼20% at pH 7 to ∼90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair. The noncanonical pair adjacent to the GA pair in can either stabilize or destabilize the loop, consistent with the sequence-dependent thermodynamics of GA pairs. No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., and ), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures. A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.
Co-reporter:Marta Carlucci, Elzbieta Kierzek, Anna Olejnik, Douglas H. Turner and Ryszard Kierzek
Biochemistry 2009 Volume 48(Issue 46) pp:
Publication Date(Web):October 16, 2009
DOI:10.1021/bi901506f
Hybridization to RNA is important for many applications, including antisense therapeutics, RNA interference, and microarray screening. Similar thermodynamic stabilities of A-U and G-U base pairs result in difficulties in selective binding to RNA. Moreover, A-U pairs are weaker than G-C pairs so that binding is sometimes weak when many A-U pairs are present. It is known, however, that replacement of uridine with 2-thiouridine significantly improves binding and selectivity. To test for additional improvement of binding and of the specificity for binding A over G, LNA-2-thiouridine was synthesized for the first time and incorporated into many LNA-2′-O-methyl-RNA/RNA duplexes. UV melting was used to measure the thermodynamic effect of replacing 2′-O-methyluridine with 2′-O-methyl-2-thiouridine or LNA-2-thiouridine. The 2-thiouridine usually enhances binding and selectivity. Selectivity is optimized when a single 2-thiouridine is placed at an internal position in a duplex.
Co-reporter:Matthew D. Disney;Jessica L. Childs
Biopolymers 2004 Volume 73(Issue 1) pp:
Publication Date(Web):11 NOV 2003
DOI:10.1002/bip.10520
RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence. © 2003 Wiley Periodicals, Inc. Biopolymers 73: 151–161, 2004
Co-reporter:Matthew D. Disney Dr.;Jessica L. Childs Dr.
ChemBioChem 2004 Volume 5(Issue 12) pp:
Publication Date(Web):8 NOV 2004
DOI:10.1002/cbic.200400159
Fungal pathogens are increasing in prevalence due to an increase in resistant strains and the number of immunocompromised humans. Candida albicans is one of these pathogens, and ∼40 % of strains contain a group I self-splicing intron, which is a potential RNA drug target, in their large subunit rRNA precursor. Here, we report that Hoechst 33258 and derivatives thereof are selective inhibitors of C. albicans group I intron self-splicing with an IC50 of 17 μM in 2 mM Mg2+. Chemical probing of the intron in the presence of Hoechst 33258 reveals that the folding of several nucleotides in the P4/P6 region of the intron is affected. A nucleotide near the J4/5 region is protected from chemical modification in the presence of Hoechst 33258 and several nearby are more reactive; this suggests that this region is the molecule's binding site. These results expand the available information on small-molecule targeting of RNA and suggest that the RNAtargeting scaffold provided by Hoechst may prove valuable in designing compounds that inhibit the functions of RNA.
Co-reporter:David H. Mathews;Jessica L. Childs;Michael Zuker;Matthew D. Disney;Susan J. Schroeder
PNAS 2004 Volume 101 (Issue 19 ) pp:7287-7292
Publication Date(Web):2004-05-11
DOI:10.1073/pnas.0401799101
A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints
determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either
adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental
results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this
method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased
from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences
with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%.
This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of
energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28%
to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification
contain on average 84% of known canonical base pairs.
Co-reporter:Matthew D. Disney;Constantine G. Haidaris
PNAS 2003 100 (4 ) pp:1530-1534
Publication Date(Web):2003-02-18
DOI:10.1073/pnas.0337462100
Candida albicans is a significant cause of disease in immunocompromised humans. Because the number of people infected by fungal pathogens
is increasing, strategies are being developed to target RNAs in fungi. This work shows that oligonucleotides can serve as
therapeutics against C. albicans. In particular, oligonucleotides are taken up from cell culture medium in an energy-dependent process. After uptake, oligonucleotides,
including RNA, remain mostly intact after 12 h in culture. For culture conditions designed for mammalian cells, intracellular
concentrations of oligonucleotides in C. albicans exceed those in COS-7 mammalian cells, suggesting that uptake can provide selective targeting of fungi over human cells.
A 19-mer 2′OMe (oligonucleotide with a 2′-O-methyl backbone) hairpin is described that inhibits growth of a C. albicans strain at pH < 4.0. This pH is easily tolerated in some parts of the body subject to C. albicans infections. In vivo dimethyl sulfate modification of ribosomal RNA and the decreased rate of protein synthesis suggest that this hairpin's activity
may be due to targeting the ribosome in a way that does not depend on base pairing. Addition of anti-C. albicans oligonucleotides to COS-7 mammalian cells has no effect on cell growth. Evidently, oligonucleotides can selectively serve
as therapeutics toward C. albicans and, presumably, other pathogens. Information from genome sequencing and functional genomics studies on C. albicans and other pathogens should allow rapid design and testing of other approaches for oligonucleotide therapies.
Co-reporter:Douglas H. Turner;Matthew D. Disney;Jessica L. Childs
PNAS 2002 Volume 99 (Issue 17 ) pp:11091-11096
Publication Date(Web):2002-08-20
DOI:10.1073/pnas.172391199
RNA is becoming an important therapeutic target. Many potential RNA targets require secondary or tertiary structure for function.
Examples include ribosomal RNAs, RNase P RNAs, mRNAs with untranslated regions that regulate translation, and group I and
group II introns. Here, a method is described to inhibit RNA function by exploiting the propensity of RNA to adopt multiple
folded states that are of similar free energy. This method, called oligonucleotide directed misfolding of RNA (ODMiR), uses
short oligonucleotides to stabilize inactive structures. The ODMiR method is demonstrated with the group I intron from Candida albicans, a human pathogen. The oligonucleotides, L(TACCTTTC) and TLCTLACLGALCGLGCLC, with L denoting a locked nucleic acid residue, inhibit 50% of group I intron splicing in a transcription mixture at about
150 and 30 nM oligonucleotide concentration, respectively. Both oligonucleotides induce misfolds as determined by native gel
electrophoresis and diethyl pyrocarbonate modification. The ODMiR approach provides a potential therapeutic strategy applicable
to RNAs with secondary or tertiary structures required for function.
Co-reporter:Susan J. Schroeder;Mark E. Burkard
Biopolymers 1999 Volume 52(Issue 4) pp:
Publication Date(Web):29 MAR 2001
DOI:10.1002/1097-0282(1999)52:4<157::AID-BIP1001>3.0.CO;2-E
The energetics of small internal loops are important for prediction of RNA secondary and tertiary structure, selection of drug target sites, and understanding RNA structure–function relationships. Hydrogen bonding, base stacking, electrostatic interactions, backbone distortion, and base-pair size compatibility all contribute to the energetics of small internal loops. Thus, the sequence dependence of these energetics are idiosyncratic. Current approximations for predicting the free energies of internal loops consider size, asymmetry, closing base pairs, and the potential to form GA, GG, or UU pairs. The database of known three-dimensional structures allows for comparison with the models used for predicting stability from sequence. © 2001 John Wiley & Sons, Inc. Biopoly (Nucleic Acid Sci) 52: 157–167, 1999/2000
Co-reporter:Elzbieta Kierzek, Shawn M. Christensen, Thomas H. Eickbush, Ryszard Kierzek, ... Walter N. Moss
Journal of Molecular Biology (17 July 2009) Volume 390(Issue 3) pp:428-442
Publication Date(Web):17 July 2009
DOI:10.1016/j.jmb.2009.04.048
Sequences from the 5′ region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for a segment of this RNA is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five conserved secondary structures are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5′ segment to a protein coding function near the 3′ end. This suggests that local regions evolved under separate functional constraints (structural, coding, or both).
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