Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.     

Conformation and dynamics of Z-DNA oligomer duplex of d[(CG)3TATA(CG)3] in solution.

The conformation and dynamics of a DNA oligomer, d[(CG)3TATA(CG)3], in 4M NaClO4 (Z-TATA 16 mer) have been studied by 1H NMR. The principal results of our investigation are: (i) at low temperature d[(CG)3TATA(CG)3] exists as duplexes in both low (0.1M NaCl) and high (4M NaClO4) ionic strength solutions; (ii) CGCGCG segments undergo the B-to-Z transition in 4M NaClO4; (iii) even in 4M NaClO4 the TATA box exhibits non-Z-structures and possesses multiple conformations which are slowly exchanging on the NMR chemical shift difference time scale; and, (iv) the Z-type structure of the CGCGCG segments induced in 4M NaClO4 is more conformationally mobile than its B-type counterpart in 0.1M NaCl on the nanosecond time scale.     

The rare crystallographic structure of d(CGCGCG)(2): the natural spermidine molecule bound to the minor groove of left-handed Z-DNA d(CGCGCG)(2) at 10 degrees C

Several crystal structure analyses of complexes of synthetic polyamine compounds, including N(1)-(2-(2-aminoethylamino))ethyl)ethane-1,2-diamine PA(222) and N(1)-(2-(2-(2-aminoethylamino)ethylamino)ethyl)ethane-1,2-diamine PA(2222), and left-handed Z-DNA d(CGCGCG)(2) have been reported. However, until now, there have been no examples of naturally occurring polyamines bound to the minor groove of the left-handed Z-DNA of d(CGCGCG)(2) molecule. We have found that spermidine, a natural polyamine, is connected to the minor groove of left-handed Z-DNA of d(CGCGCG)(2) molecule in a crystalline complex grown at 10 degrees C. The electron density of the DNA molecule was clear enough to determine that the spermidine was connected in the minor groove of two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2). This is the first example that a spermidine molecule can form a bridge conformation between two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2) in the minor groove.     

Conformational analysis of r(CGCGCG) in aqueous solution: an A-type double helical conformation studied by two-dimensional nuclear Overhauser effect spectroscopy.

The conformation of the hexanucleoside pentaphosphate r( CGCGCG ) in aqueous solution was studied by circular dichroism, 1H- and 31P-NMR spectroscopy. The base-, H1'- and H2'-proton resonances were assigned by means of 2D-NOE spectroscopy. The base- and H1'-proton chemical shifts were studied as a function of temperature. Proton-proton distances are computed in A- and A'-RNA as well as in A-, B- and Z-DNA. A qualitative interpretation of the observed 2D-NOE intensities shows that r( CGCGCG ) adopts a regular A-type double helical conformation under our experimental conditions. The CD- and 31P-NMR experiments described in this paper are in agreement with this structure both under low- and high-salt conditions.     

Double-helix formation of self-complementary chimeric oligonucleotides r(CG)nd(CG)3-n (n = 0, 1, 2, 3).

Double-helix formations of self-complementary chimeric hexanucleotides, r(CGCGCG), r(CGCG)d(CG), r(CG)d(CGCG), and d(CGCGCG), have been studied spectrophotometrically an thermodynamically in 1 mol dm-3 NaCl buffer. CD (circular dichroism) spectra showed that r(CGCGCG), r(CGCG)d(CG), and r(CG)d(CGCG) formed A-type double helix, while d(CGCGCG) formed B-type double helix. The stabilization energies of these helices at 37 degrees C obtained from UV melting analyses were 9.2 kcal mol-1 for r(CGCGCG), 8.2 kcal mol-1 for r(CGCG)d(CG), 6.8 kcal mol-1 for r(CG)d(CGCG), and 8.5 kcal mol-1 for d(CGCGCG), respectively.     

Effect of the G.T mismatch on backbone and sugar conformations of Z-DNA and B-DNA: analysis by Raman spectroscopy of crystal and solution structures of d(CGCGTG) and d(CGCGCG)

The Z-DNA crystal structures of d(CGCGTG) and d(CGCGCG) are compared by laser Raman spectroscopy. Raman bands originating from vibrations of the phosphodiester groups and sensitive to the DNA backbone conformation are similar for the two structures, indicating no significant perturbation to the Z-DNA backbone as a result of the incorporation of G.T mismatches. Both Z structures also exhibit Raman markers at 625 and 670 cm-1, assigned respectively to C3'-endo/syn-dG (internal) and C2'-endo/syn-dG conformers (3' terminus). Additional Raman intensity near 620 and 670 cm-1 in the spectrum of the d(CGCGTG) crystal is assigned to C4'-exo/syn-dG conformers at the mismatch sites (penultimate from the 5' terminus). A Raman band at 1680 cm-1, detected only in the d(CGCGTG) crystal, is assigned to the hydrogen-bonded dT residues and is proposed as a definitive marker of the Z-DNA wobble G.T pair. For aqueous solutions, the Raman spectra of d(CGCGTG) and d(CGCGCG) are those of B-DNA, but with significant differences between them. For example, the usual B-form marker band at 832 cm-1 in the spectrum of d(CGCGTG) is about 40% less intense than the corresponding band in the spectrum of d(CGCGCG), and the former structure exhibits a companion band at 864 cm-1 not observed for d(CGCGCG). The simplest interpretation of these results is that the conventional B-form OPO geometry occurs for only 6 of the 10 OPO groups of d(CGCGTG). The remaining four OPO groups, believed to be those at or near the mismatch site, are in an "unusual B" conformation which generates the 864 cm-1 band.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of d(GCGCGCG) with 5''-overhang G residues.

The crystal structure of the DNA heptamer d(GCGCGCG) has been solved at 1.65 A resolution by the molecular replacement method and refined to an R-value of 0.184 for 3598 reflections. The heptamer forms a Z-DNA d(CGCGCG)2 with 5'-overhang G residues instead of an A-DNA d(GCGCGC)2 with 3'-overhang G residues. The overhang G residues from parallel strands of two adjacent duplexes form a trans reverse Hoogsteen G x G basepair that stacks on the six Z-DNA basepairs to produce a pseudocontinuous helix. The reverse Hoogsteen G x G basepair is unusual in that the displacement of one G base relative to the other allows them to participate in a bifurcated (G1)N2 . . . N7(G8) and an enhanced (G8)C8-H . . . O6(G1) hydrogen bond, in addition to the two usual hydrogen bonds. The 5'-overhang G residues are anti and C2'-endo while the 3'-terminal G residues are syn and C2'-endo. The conformations of both G residues are different from the syn/C3'-endo for the guanosine in a standard Z-DNA. The two cobalt hexammine ions bind to the phosphate groups in both GpC and CpG steps in Z(I) and Z(II) conformations. The water structure motif is similar to the other Z-DNA structures.     

High salt solution structure of a left-handed RNA double helix

Right-handed RNA duplexes of (CG)_n sequence undergo salt-induced helicity reversal, forming left-handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)₂ determined in 6 M NaClO₄. This is the first nucleic acid motif determined at such high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6_(n)-H5′/H5″_(n)-H8_(n+1)-H1′_(n+1)-H6_(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4–7°), whereas GpC steps have large negative values (−61°). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2′-OH groups, and the major groove are well defined. The 2′-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2′-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.     

Salt induced conformational transition between A and Z forms of r(CGCGCG) as revealed by a Raman spectroscopic study

Solution conformation in different conditions of r(CGCGCG) has been studied by a Raman spectroscopic method. In NaCl solution, r (CGCGCG) takes only an A-form duplex in which guanosine and cytidine have C3'endo-anti conformation even at 5M salt concentration. In much higher ionic strength condition (5M NaCl plus 1M MgCl2 or 6M NaClO4), it undergoes a transition to a left-handed Z-form. The Raman spectrum of the Z-form RNA was found to be very similar to that of Z-form DNA, suggesting that Z-RNA involves a C3'endo-syn guanosine and an in between form of C2'endo-Cl'exo-anti cytidine.     

Synthesis and hybridization studies of oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine (2'-alpha-hm-dT)

We report the first investigation of oligoribonucleotides containing a few 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta-D-ribofuranosyl)thymine units (or 2'-hm-dT, abbreviated in this work as 'H'). Both the 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. The hybridization properties of the modified oligonucleotides have been studied via thermal denaturation and circular dichroism studies. While 3',5'-linked H was shown previously to significantly destabilize DNA:RNA hybrids and DNA:DNA duplexes (modification in the DNA strand; DeltaT(m) approximately -3 degrees C/insert), we find that 2',5'-linked H have a smaller effect on 2',5'-RNA:RNA and RNA:RNA duplexes (DeltaT(m) = -0.3 degrees C and -1.2 degrees C, respectively). The incorporation of 3',5'-linked H into 2',5'-RNA:RNA and RNA:RNA duplexes was found to be more destabilizing (-0.7 degrees C and -3.6 degrees C, respectively). Significantly, however, the 2',5'-linked H units confer marked stability to RNA hairpins when they are incorporated into a 2',5'-linked tetraloop structure (DeltaT(m) = +1.5 degrees C/insert). These results are rationalized in terms of the compact and extended conformations of nucleotides.     

Internal mobility of the ologonucleotide duplexes d(TCGCG)2 and d(CGCGCG)2 in aqueous solution from molecular dynamics simulations

Summary In this paper we present longitudinal relaxation times, order parameters and effective correlation times for the base and sugar carbons in both strands of the oligonucleotide duplexes d(TCGCG)₂ and d(CGCGCG)₂, as calculated from 400 ps molecular dynamics trajectories in aqueous solution. The model-free approach (Lipari and Szabo, 1982) was used to determine the amplitudes and time scales of the internal motion. Comparisons were made with NMR relaxation measurements (Borer et al., 1994). The order parameters could acceptably be reproduced, and the effective correlation times were found to be lower than the experimental estimates. Reasonable T₁ relaxation times were obtained in comparison with experiment for the nonterminal nucleosides. The T₁ relaxation times were found to depend mainly on the order parameters and overall rotational correlation time.Abbreviations MD molecular dynamics - CSA chemical shift anisotropy To whom correspondence should be addressed.     

Interaction between the Z-type DNA duplex and 1,3-propanediamine: crystal structure of d(CACGTG)2 at 1.2 A resolution

The crystal structure of a hexamer duplex d(CACGTG)(2) has been determined and refined to an R-factor of 18.3% using X-ray data up to 1.2 A resolution. The sequence crystallizes as a left-handed Z-form double helix with Watson-Crick base pairing. There is one hexamer duplex, a spermine molecule, 71 water molecules, and an unexpected diamine (Z-5, 1,3-propanediamine, C(3)H(10)N(2)) in the asymmetric unit. This is the high-resolution non-disordered structure of a Z-DNA hexamer containing two AT base pairs in the interior of a duplex with no modifications such as bromination or methylation on cytosine bases. This structure does not possess multivalent cations such as cobalt hexaammine that are known to stabilize Z-DNA. The overall duplex structure and its crystal interactions are similar to those of the pure-spermine form of the d(CGCGCG)(2) structure. The spine of hydration in the minor groove is intact except in the vicinity of the T5A8 base pair. The binding of the Z-5 molecule in the minor grove of the d(CACGTG)(2) duplex appears to have a profound effect in conferring stability to a Z-DNA conformation via electrostatic complementarity and hydrogen bonding interactions. The successive base stacking geometry in d(CACGTG)(2) is similar to the corresponding steps in d(CG)(3). These results suggest that specific polyamines such as Z-5 could serve as powerful inducers of Z-type conformation in unmodified DNA sequences with AT base pairs. This structure provides a molecular basis for stabilizing AT base pairs incorporated into an alternating d(CG) sequence.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove

The crystal and molecular structure of 2′-O-Me(CGCGCG)₂ has been determined at 1.19 Å resolution, at 100 K, using synchrotron radiation. The structure in space group P3₂12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3′-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2′-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O⁶ and N⁷ of guanosine and N⁴ of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2′-O-Me(CGCGCG)₂ structure in the P3₂12 and P6₁22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2′-O-Me(CGCGCG)₂ is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.     

The effect of methylation of the 6 oxygen of guanine on the structure and stability of double helical DNA

The effect of methylation of the O-6 position of guanine in short segments of double helical DNA has been investigated by molecular mechanical simulations on the sequences d(CGCGCG)2, d(CGC[OMG]CG)2, d(CGT[OMG]CG)2, d(CGC[OMC]CG/(CGCGCG), d(CGC[OMG]CG/d(CGTGCG), d(CGCGAATTCGCG)2 and d(CGCGAATTC[OMG]CG)2. Guanines methylated at the O-6 position are found to form hydrogen bonds of roughly equal strength to cytosine and thymine. The optimum structure of these modified base pairs are not dramatically different from normal GC pairs, but both involve some bifurcation of the proton donors of cytosine (4NH2) or thymine (3NH) between the guanine N3 and O6 groups.     

Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures.

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1', H2',2', H3', and H4' protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 M-NaCl, 10 mM-sodium phosphate, 2H2O solution at 0 degree C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1' protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7.45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8.0 to 8.3 p.p.m.) in an anti alignment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Water and ion binding around RNA and DNA (C,G) oligomers

The dynamics, hydration, and ion-binding features of two duplexes, the A(r(CG)(12)) and the B(d(CG)(12)), in a neutralizing aqueous environment with 0.25 M added KCl have been investigated by molecular dynamics (MD) simulations. The regular repeats of the same C=G base-pair motif have been exploited as a statistical alternative to long MD simulations in order to extend the sampling of the conformational space. The trajectories demonstrate the larger flexibility of DNA compared to RNA helices. This flexibility results in less well defined hydration patterns around the DNA than around the RNA backbone atoms. Yet, 22 hydration sites are clearly characterized around both nucleic acid structures. With additional results from MD simulations, the following hydration scale for C=G pairs can be deduced: A-DNA相似文献   

Study of self-association of molecules of deoxyhexanucleotides 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in water solutions by NMR

The self-association of deoxyribohexanucleoside pentaphosphates 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in aqueous salt solutions was studied by 1 D- and 2 D homonuclear PMR and heteronuclear 1H-31P-spectroscopies. Signals from nonexchangeable protons of hexamers in NMR spectra were assigned using the available 2M-TOCSY, 2M-NOESY, and 1H-31P-(HMBS) spectra. The dependences of proton chemical shifts of deoxyhexanucleotides on concentration and temperature were measured. In terms of the two-states model (monomer-duplex), constants and thermodynamic parameters of self-association of hexamer molecules in solution were obtained based on these dependences. The values obtained correlate well with theoretical values calculated using the model of the "nearest neighbor" for the formation of duplexes of sequences d(CGTACG) and d(CGCGCG).     

Hydration pattern of A4T4 and T4A4 DNA: a molecular dynamics study

Hydration pattern and energetics of 'A-tract' containing duplexes have been studied using molecular dynamics on 12-mer self-complementary sequences 5'-d(GCA4T4GC)-3' and 5'-d(CGT4A4CG)-3'. The structural features for the simulated duplexes showed correlation with the corresponding experimental structures. Analysis of the hydration pattern confirmed that water network around the simulated duplexes is more conformation specific rather than sequence specific. The calculated heat capacity change upon duplex formation showed that the process is entropically driven for both the sequences. Furthermore, the theoretical free energy estimates calculated using MMPBSA approach showed a higher net electrostatic contribution for A4T4 duplex formation than for T4A4, however, energetically both the duplexes are observed to be equally stable.     

Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).