A-form Duplex Research Articles

Solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at the center.

The solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at its center, (ggagaugac).(GmUmCmATCTCmCm), where lowercase letters, capital letters, and capital letters with the subscript m are RNA, DNA, and 2'-O-methylated RNA residues, respectively, was determined by observing the NMR spectra and performing the full relaxation matrix refinement. The 2'-O-methylation gives several characteristic features to oligoribonucleotides. In addition, this hybrid duplex is cleaved at a specific position by Escherichia coli ribonuclease HI, and so the role of the tertiary structure during the substrate recognition by the enzyme is of interest. The NOE connectivities among the proton resonances revealed that the duplex was a right handed helix. The 2'-O-methylated RNA segments had a typical C3'-endo conformation, and the 2'-O-methyl groups were directed to the minor groove of this duplex, taking the torsion angles phi (C1'-C2'-02'-CH3) that were all gauche(+). The DNA residues in the central RNA.DNA hybrid duplex formed the C3'-endo conformation, except for the middle thymine residue. No remarkable structural discontinuities were observed around the junction sites at either the 5'- or 3'-end of the DNA. The overall structure was close to the typical A-form duplex.

Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone.

Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids.

In this study we report for the first time the binding affinity of RNase H1 for oligonucleotide duplexes. We used a previously described 17-mer antisense sequence [Monia, B. P., Johnston, J. F., Ecker, D. J., Zounes, M. A., Lima, W. F., & Freier, S. M. (1992) J. Biol. Chem. 267, 19954-19962] hybridized to a complementary oligoribonucleotide to evaluate both the binding affinity and the catalytic rate of RNase H1. The dissociation constants (Kd) of RNase H1 for the various substrates tested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes. Catalytic rates were determined using heteroduplex substrates containing chimeric antisense oligonucleotides composed of a five-base deoxynucleotide sequence flanked on either side by chemically modified nucleotides. We find that the enzyme preferentially binds A-form duplexes: RNase H bound A-form duplexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-strand oligonucleotides. The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes containing various 2'-sugar modifications, while the cleavage rates for these chemically modified substrates were without exception slower than for the wild type substrate. The introduction of a single positively charged 2'-propoxyamine modification into the chimeric antisense oligonucleotide portion of the heteroduplex substrate resulted in both decreased binding affinity and a slower rate of catalysis by RNase H. The cleavage rates for heteroduplexes containing single-base mismatch sequences within the chimeric oligonucleotide portion varied depending on the position of the mismatch but had no effect on the binding affinity of the enzyme. These results offer further insights into the physical binding properties of the RNase H-substrate interaction as well as the design of effective antisense oligonucleotides.

Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage.

Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available. We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2'-oxygen in line with the adjacent phosphate on the 3' side, where it is poised for nucleophilic attack. Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites.

Design and analysis of RNA structure-specific agents as potential antivirals

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants > 10(7)M-1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

Design and Analysis of RNA Structure‐Specific Agents as Potential Antivirals

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants >107M−1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

Design and analysis of RNA structure-specific agents as potential antivirals.

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants > 10(7)M-1), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.

RNA hydration: a detailed look.

The crystal structure of the RNA duplex [r(CCCCGGGG)]2 has been refined to 1.46 A resolution with room temperature synchrotron diffraction data. This represents the highest resolution reported to date for an all-RNA oligonucleotide and is well beyond the best resolution ever achieved with an A-form DNA duplex. The analysis of the ordered hydration around the octamer duplex reveals conserved regular arrangements of water molecules in both grooves. In the major groove, all located first shell water molecules can be fitted into a pattern that is repeated through all eight base pairs, involves half the phosphate oxygens, and joins the two strands. In the minor groove, roughly across its narrowest dimension, tandem water molecules link the 2'-hydroxyl groups of adjacent nucleotides in base-pair steps in a similarly regular fashion. The structure provides evidence for an important role of the 2'-hydroxyl groups in the thermodynamic stabilization of RNA, beyond their known functions of locking the sugar pucker and mediating 3' --> 5' intrastrand O2'...O4' hydrogen bonds. The ribose 2'-hydroxyls lay the foundation for the enthalpic stability of the RNA relative to the DNA duplex, both as a scaffold for the water network in the minor groove and through their extensive individual hydration.

Structure of chimeric duplex junctions: solution conformation of the retroviral Okazaki-like fragment r(ccca)d(AATGA).d(TCATTTGGG) from Moloney murine leukemia virus.

We have determined the solution structure of the synthetic chimeric duplex r(ccca)d(AATGA).d(TCATTTGGG) by two-dimensional NMR, distance geometry, restrained molecular dynamics, and full relaxation matrix simulation of the two-dimensional nuclear Overhauser effect spectra at various mixing times. The chimeric strand of this duplex consists of the last four residues of the tRNA(Pro) primer for (-) strand DNA synthesis of Moloney murine leukemia virus and the first five residues of the (-) strand DNA produced by extending this primer; the complementary DNA strand corresponds to the (+) strand product from this template. The hybrid section of this chimeric duplex assumes a structure similar to that found for pure hybrid duplexes of mixed sequence, while the DNA section assumes a conformation closer to B-form DNA. There is significant distortion of the duplex at the hybrid-DNA junction which is manifested in marked changes in the helical parameters buckle, roll, and tip, changes in glycosidic torsion angles, and changes in the backbone torsion angles delta, epsilon, and zeta. The sugar conformations also undergo large changes, from heteromerous puckers in the hybrid section to a more B-form in the DNA section. Furthermore, the intrastrand phosphate separation in the chimeric strand is more typical of A-form duplexes in the RNA section but more like B-form duplexes in the DNA section. In the DNA section the minor groove width changes gradually from B-form at the periphery and approaches hybrid-like dimensions closer to the junction. The structural discontinuities act synergistically to produce a bend of 18 +/- 3 degrees at the junction. The global structure of this sequence is similar to that previously found in the chemically analogous Okazaki fragment r(gcg)d(TATACCC).d(GGGTATACGC) in solution. Such structure homology suggests a possible link between structure and function with respect to the recognition and cleavage of the junction RNA residues in both retroviral chimeras and Okazaki fragments during reverse transcription and normal DNA replication.

NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures.

Software packages NUPARM and NUCGEN, are described, which can be used to understand sequence directed structural variations in nucleic acids, by analysis and generation of non-uniform structures. A set of local inter basepair parameters (viz. tilt, roll, twist, shift, slide and rise) have been defined, which use geometry and coordinates of two successive basepairs only and can be used to generate polymeric structures with varying geometries for each of the 16 possible dinucleotide steps. Intra basepair parameters, propeller, buckle, opening and the C6...C8 distance can also be varied, if required, while the sugar phosphate backbone atoms are fixed in some standard conformation in each of the nucleotides. NUPARM can be used to analyse both DNA and RNA structures, with single as well as double stranded helices. The NUCGEN software generates double helical models with the backbone fixed in B-form DNA, but with appropriate modifications in the input data, it can also generate A-form DNA and RNA duplex structures.

Synthesis of 5'-O-(4,4'-Dimethoxytrityl)-2'-O-(tert-butyldimethylsilyl)-5-fluorouridine 3'-(Cyanoethyl N,N-diisopropylphosphoramidite) and Its Use in the Synthesis of RNA

5-Fluorouridine (FUrd) has been successfully converted to 5'-O-(4,4'-dimethoxytrityl)-2'-O-(tertbutyldimethylsilyl)-5-fluorouridine 3'-(cyanoethyl N,N-diisopropyl(phosphoramidite) by methods similar to those employed for the preparation of other ribonucleoside phosphoramidites. The desired product, as well as its precursors and the incorrect regioisomers, are prepared and fully characterized by 1 H NMR, 19 F NMR, 31 P NMR, FAB-MS, UV absorbance, elemental analysis, and melting points. The purified material is used for the production of RNA by solid-phase methods. Coupling yields of 94% are obtained with this material. The resulting RNA is readily purified and used for biophysical studies. The self-complemetary RNA decamer 5'-rGCGAAU(FU)CGC is prepared and purified. The material adopts an A-form duplex in solution at neutral pH as characterized by CD spectroscopy. The thermal stability of the duplex is similar to the parent duplex oligoribonucleotide prepared with uridine instead of 5-fluorouridine. The control duplex has a T m of 53.8 and the FUrd substituted duplex has a T m of 56.6 ×b0° C as determined by UV hyperchromicity at 260nm. The CD spectra and UV hyperchromicity data for the duplex oligoribonucleotide containing FUrd have a slight pH dependence indicating that ionization of FUrd has a slight but measurable impact on RNA duplex stability. 1D NMR spectroscopy of the imino hydrogens for this duplex in H 2 O solution confirms the formation of Watson-Crick base pairs. The imino hydrogen resonance for the FUrd-A base pair is moved downfield and broadened compared with a control duplex oligoribonucleotide as a consequence of the electron-withdrawing inductive effect of fluorine

Structure of a DNA : RNA Hybrid Duplex: Why RNase H Does Not Cleave Pure RNA

The solution structure of the DNA : RNA hybrid duplex d(GTCACATG) : r(caugugac) has been determined by means of two-dimensional nuclear Overhauser effect (2D-NOE) spectra, restrained molecular dynamics and full-relaxation matrix stimulation of the 2D-NOE spectra. The DNA : RNA hybrid duplex assumes neither an A-form nor a B-form structure in solution, but an intermediate heteromerous duplex structure. The sugars of the RNA strand have a normal N-type C3′- endo conformation, but the DNA strand sugars have neither N-type nor S-type conformations; instead, they have an unexpected intermediate O4′- endo conformation. The negative x -displacement, as well as the small rise and positive inclination of the base-pairs, resembles A-form morphology but the minor groove width is intermediate between that of A-form and B-form duplexes. Both the DNA and RNA strands show prominent sequence-dependent variations in their helical parameters. Combined analysis of NOE and J-coupling data indicates that the DNA sugars are not in a dynamical two-state equilibrium. The detailed three-dimensional structure of this DNA : RNA hybrid molecule leads to a proposed model for its interaction with RNase H. Several specific structural features of the enzyme complexed with the hybrid duplex appear to explain the mechanism whereby RNase H discriminates between DNA : RNA hybrid duplexes and pure RNA : RNA duplexes.

NMR spectroscopy of DNA/RNA hybrids

RNA/DNA hybrids are formed in many biological processes. Earlier work on hybrid molecules had indicated that although their stability varied according to their sequence, all these duplexes were apparently A-form. Recent structural experiments have been directed toward defining how these hybrid A-form conformations differ from canonical RNA A-form duplexes.

High-resolution NMR study of a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein, ricin.

Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Its substrate domain forms a double helical stem and a 17-base loop that includes the sequence GAGA, the second adenosine of which corresponds to A4324. Recently, studies of mutant RNAs have shown that the four-nucleotide loop, GAGA, can function as a substrate for ricin. To investigate the structure that is recognized by ricin, we studied the properties of a short synthetic substrate, the dodecaribonucleotide r-CUCAGAGAUGAG, which forms a RNA hairpin structure with a GABA loop and a stem of four base pairs. The results of NMR spectroscopy allowed us to construct the solution structure of this oligonucleotide by restrained molecular-dynamic calculations. We found that the stem region exists as an A-form duplex. 5G and 8A in the loop region form an unusual G:A base pair, and the phosphodiester backbone has a turn between 5G and 6A. This turn seems to help ricin to gain access to 6A which is the only site of depurination in the entire structure. The overall structure of the GAGA loop is similar to those of the GAAA and GCAA loops that have been described but that are not recognized by ricin. Therefore, in addition to the adenosine at the depurination site, the neighboring guanosine on the 3' side (7G) may also play a role in the recognition mechanism together with 5G and 8A.

RNA-protein interactions: Current Opinion in Structural Biology 1992, 2: 131…-137

Recent biochemical experiments have revealed the secondary structural elements of RNA that are recognized by various RNA-binding proteins. These include structures such as hairpins, bulges, bubbles and pseudoknots that are distinct from the double-stranded form. Proteins may recognize irregular backbone conformations and interact specifically with bases in these regions as these are more exposed than those in the A-form RNA duplex. The ribonucleoprotein domain is one of the most important RNA-binding modules and is found in many RNA-binding proteins. The recent crystal structure of the U1 A small nuclear ribonucleoprotein in conjunction with biochemical experiments shows how this domain may interact with an RNA hairpin.

Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.

A Note on Crystal Packing and Global Helix Structure in Short A-DNA Duplexes

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.

Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study

A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.