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R H Sarma

Publications and source records attributed to R H Sarma.

At least 19 recordsLinked to original sources
PubMed1997

GC rich DNA oligonucleotides with narrow minor groove width.

Investigation of the width of the minor groove using 500 MHz NMR spectroscopy in three closely related 11-mer B-DNA duplexes shows that the minor groove is narrow in a GC rich oligonucleotide, and that a narrow minor groove is not something endemic to DNAs with persistent repetitions of adenine nucleotides (A-tract DNA). The width of the groove is dictated by local sequence contexts and independent of neighboring A-tract DNA.

Base Composition↗
PubMed1997

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

Catalysis↗
PubMed1995

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Base Sequence↗
PubMed1995

Modeling of a possible conformational change associated with the catalytic mechanism in the hammerhead ribozyme.

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

Base Sequence↗
PubMed1993

Decreased interstrand H2-H1' distance in the GC-rich part of the duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) in solution at low temperature: proton nuclear magnetic resonance investigation.

The non-self-complementary undecadeoxyribonucleotide duplex d(CCTCAAACTCC).d(GGAGTTTGAGG) was studied by one- and two-dimensional NMR methods in solution at low and room temperatures. The width of the minor groove of the duplex was determined on the basis of the NOE's between adenine's H2 protons and H1' protons from the complementary strand. In agreement with the previous reports, we found that the A3.T3 block forms a structure with a narrow minor groove at 5 degrees C, with the H2-H1' interstrand distance decreasing in the 5'-to-3' direction along the strand of adenines. Surprisingly, this distance is still short in the GC-rich part of the duplex downstream from the A-tract. This finding is interpreted in terms of pronounced buckle angles in the oligo(purine).oligo(pyrimidine) blocks, which diminish the H2-H1' interstrand distances. Both CA(n)- and AnC- junctions have distinct patterns of the proton chemical shifts, which suggests that both junctions may have some specific conformations in solution. Also, we report the temperature-driven changes in proton chemical shifts, which are significant in all parts of the duplex, except the 5'-ends of both strands. The structural interpretation for these changes is proposed, on the basis of the following notion: at low temperature the narrow minor groove is formed between the central A3.T3 trimer and the 3'-ends of both DNA strands, while the 5'-ends remain relatively exposed to the solvent.

Adenine↗
PubMed1992

NMR study of self-paired parallel duplex of d(AAAAACCCCC) in solution.

The oligonucleotide d(A5C5) in solution forms a parallel self-duplex at neutral and low pH values. H2O NMR spectra at pH 5.1 indicate the presence of five imino resonances at lower temperatures; and the structure is stable up to 60 degrees C. These signals can arise only from the hemiprotonated C+.C pairs [Westhof, E. and Sundaralingham, M. (1980) Biochemistry 77, 1852-1856; Westhof, E. and Sundaralingham, M. (1980) J. Mol. Biol. 142, 331-361] and constitute the first direct observation of C+.C hemiprotonated pairs in solution. The cross peaks from H1's and more than five distinct AH8's in 500 MHz 1H 2D-NOESY spectra indicate that there are two conformationally different and energetically similar A-tracts. There is good qualitative agreement between NOESY data and two theoretically derived structures in which A-tracts are reverse Watson-Crick and reverse Hoogsteen base-paired, respectively.

Base Composition↗
PubMed1992

Systematic study of nuclear Overhauser effects vis-à-vis local helical parameters, sugar puckers, and glycosidic torsions in B DNA: insensitivity of NOE to local transitions in B DNA oligonucleotides due to internal structural compensations.

A method has been developed to solve structures of DNA oligomers in solution from the experimental NOE data. The method is a combination of two approaches: (1) full matrix NOESY simulations and (2) conformational calculations of DNA double helix based on generalized helical parameters. The process of the refinement of a solution structure does not involve NMR-derived interproton distance constraints; rather it consists of a direct fitting of a structure to the experimental NOE data, a weighted sum of energy, and R factor being under minimization. A helical parameters-based generation of DNA forms makes it possible to organize the search for the optimal structure more effectively, systematically varying starting conformations. The method has been used to calculate a structure for the self-complementary DNA hexamer GGATCC, which is consistent with the available experimental data. The structure belongs to the B family of forms, although the local structural heterogeneity is very strong. Sugar puckers vary from O4'-exo to C3'-exo; helical steps are open with different magnitudes toward the minor groove. Next, we have addressed the question of how uniquely the structure is defined by the existing NMR data. Different structural parameters have been systematically varied, and their effect on individual NOE's and the R factor has been studied. Two energetically conjugated parameters, sugar puckers and glycosidic angles, can be determined very reliably, because of the strong dependences of the intraresidue H6/H8 to H2'/H2''/H3' NOE's. In contrast, the local helical conformation of DNA and the geometry of base pairs proved to be underdetermined by the existing NOE information, because the effect of any helical parameter on interproton distances can be compensated by the concerted changes in other parameters.

Base Composition↗
PubMed1992

Tetraplex formation of d(GGGGGTTTTT): 1H NMR study in solution.

The oligonucleotide d(G5T5) can in principle form a fully matched duplex with G.T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A5C5) at low temperature, d(G5T5) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G5T5) and d(A5C5) are mixed together in equimolar amounts and heated for several minutes at 85 degrees C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz 1H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.

Base Sequence↗
PubMed1990

Effect of the methyl group on DNA bending and curvature: structure of d(GA4U4C)2 in solution.

NMR studies on d(GA4T4C)2 and d(GT4A4C)2 indicated two important factors that contribute to intrinsic DNA bending in polymers containing A/T tracts [Sarma, M.H., et al. (1988) Biochemistry 27, 3423; Gupta, G., et al. (1988) Biochemistry 27, 7909]. They are (i) propeller-twisted A.T pairs with associated bifurcated H bonds inside the A/T tract and (ii) the base sequence that joins the two neighboring A/T tracts. As an extension of our bending project, we carried out quantitative NMR studies on the decamer d(GA4U4C)2, a structural analogue of d(GA4T4C)2, to examine the effect of the methyl group on DNA bending. On the basis of quantitative NMR analysis, we arrive at the following results. (i) The decamer d(GA4U4C)2 adopts the gross morphology of a right-handed B-DNA duplex with A and U nucleotides belonging to C2'-endo,anti domain. (ii) A.U pairs are propeller twisted and hence can result in an array of interstrand bifurcated H bonds involving N6 of A and O4 of U (one base pair apart) inside the A/U tract. (iii) The orientations of A and U with respect to the long axis of the molecule are different; as a result, at the A5-U6 sequence that joins the two A/U tracts, two neighboring frames of reference do not exactly coincide in space and a junction is created at A5-U6. (iv) Inside the A/U tract, intrastrand stacking is more compact (average separation between secessive base planes being 3.2 A) than at the A5-U6 junction, where average separation between the base planes of A5 and U6 is 3.6 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1990

Quantitative structure of a complex between a minor-groove-specific drug and a bent DNA decamer duplex: use of 2D NMR data and NOESY constrained energy minimization.

Two-dimensional nuclear magnetic resonance (2D NMR) studies on d(GA4T4C)2 and d(GT4A4C)2 [Sarma, M.H., et al. (1988) Biochemistry 27, 3423-3432; Gupta G., et al. (1988) Biochemistry 27, 7909-7919] showed that A.T pairs are propeller twisted. As a result, A/T tracts form a straight rigid structural block with an array of bifurcated inter base pair H bonds in the major groove. It was demonstrated (previous paper) that replacement of methyl group by hydrogen (changing from T to U) in the major groove does not disrupt the array of bifurcated H bonds in the major groove. In this article, we summarize results of 2D NMR and molecular mechanic studies on the effect of a minor-groove-binding A.T-specific drug on the structure d(GA4T4C)2. A distamycin analogue (Dst2) was used for this study. It is shown that Dst2 binds to the minor groove of d(GA4T4C)2 mainly driven by van der Waals interaction between A.T pairs and the drug; as a consequence, an array of bifurcated H bonds can be formed in the minor groove between amide/amino protons of Dst2 and A.T pairs of DNA. NOESY data suggest that Dst2 predominantly binds at the central 5 A.T pairs. NOESY data also reveal that, upon drug binding, d(GA4T4C)2 does not undergo any significant change in conformation from the free state; i.e., propeller-twisted A.T pairs are still present in DNA and hence the array of bifurcated H bonds must be preserved in the major groove. NOESY data for the A5-T6 sequence also indicate that there is little change in junction stereochemistry upon drug binding.

Base Composition↗
PubMed1990

In search of a Hoogsteen base paired DNA duplex in aqueous solution.

When the oligodeoxynucleotides d(A)6 and d(T)6 are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC in equilibrium with H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)6. We show that (i) addition of extra d(A)6 does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix-drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, 31P NMR spectra, 1H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded. Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.

Adenine↗
PubMed1988

On the question of DNA bending: two-dimensional NMR studies on d(GTTTTAAAAC)2 in solution.

It is very well documented that the presence of an An.Tn tract causes intrinsic DNA bending. Hagerman demonstrated that the sequence in which the An.Tn tracts are joined plays a very crucial role in determining DNA bending. For example, Hagerman showed that the polymer with a repeat of d(GA4T4C)n greater than or equal to 10 is bent but the polymer with a repeat of d(GT4A4C)n greater than or equal to 10 is not bent [Hagerman, P. J. (1986) Nature (London) 326, 720-722]. Earlier we have shown that the decamer repeat d(GA4T4C)2 is itself bent with a finite structural discontinuity at the A----T sequence [Sarma, M. H., Gupta, G., & Sarma, R. H. (1988) Biochemistry 27, 3423-3432]. In the present article, we summarize our studies on the decamer repeat d(GT4A4C)2 structure in solution. By employment of 1D and 2D 1H NMR studies at 500 MHz a complete sequential assignment has been made for the exchangeable and nonexchangeable protons belonging to the ten nucleotides. NOESY data were collected for d(GT4A4C)2 at 17 degrees C in D2O for three mixing times, 150, 100, and 50 ms. A quantitative NOESY simulation technique was employed to arrive at a structural model of d(GT4A4C)2 in solution. Our detailed analyses revealed the following structural features: (i) The duplex adopts the gross morphology of a B-DNA. (ii) All the A.T pairs are propeller twisted (less than or equal to -15 degrees). (iii) Although both A and T nucleotides belong to the C2'-endo,anticonformational domain, there is a mild variation in the actual conformation of the A and T residues. (iv) Even though there is a subtle conformational difference in the A and T nucleotides, two structural frames of T4.A4 segments are joined at the T----A sequence in such a way that there is no finite discontinuity at the junction; i.e., two neighboring frames exactly coincide at the T----A junction. Thus, our studies on d(GA4T4C)2 (Sarma et al., 1988) and on d(GT4A4C)2 (this article) reveal the structural peculiarity of the An.Tn tract and the effect of A----T/T----A sequence in causing DNA bending.

Base Composition↗
PubMed1988

Structure of a bent DNA: two-dimensional NMR studies on d(GAAAATTTTC)2.

Intrinsic DNA bending is caused by specific DNA sequences. The decamer d(GA4T4C)2, when it repeats in a synthetic polymer or in kinetoplast DNA, results in a macroscopic bending of the molecule as a whole. We employed high-resolution two-dimensional NMR methods to examine the intrinsic structural properties of the d(GA4T4C)2 duplex in solution. Examination of the NOESY data at 50- and 100-ms mixing times indicated that the kinds of observed NOEs can originate if each of the ten nucleotidyl residues belongs to the B-DNA family, i.e., C2'-endo,anti. However, the degree of observed NOE intensities from the A-T junction as well as the observed AH2-AH2 cross-peaks from adjacent AT pairs could not be rationalized on the basis of a straight B-DNA model but could be explained by only a B-DNA model with some structural discontinuity at the A-T junction--the site of 2-fold symmetry in the molecule. In view of the fact that the degree of observed NOE intensities can be complicated by spin diffusion and by fine structural distortion, we have resorted to the use of quantitative theoretical NOESY simulation (which takes into account primary, secondary, and higher orders of NOE) to delineate the structural discontinuity at the A-T junction and to arrive at a structure for the duplex d(GA4T4C)2. We propose a "junction B-DNA model" which can quantitatively explain the 2D NOESY data at 100- and 50-ms mixing times. In this model the two structural blocks in the molecule, i.e., d(GA4).d(T4C) and d(T4C).d(GA4), are conformationally equivalent and are connected at the A-T junction where the base pairs are stably stacked, but the two local structural frames do not coincide in space. This model can create an overall bending of 10 degrees with a center of curvature 50 A away from the center of the duplex. It is the thesis of this paper that the observed bending in polymers with a repeat of d(GA4T4C)2 and the bending in natural DNAs where AnTn.AnTn repeats are present originate at the oligonucleotide repeat level.

Base Sequence↗
PubMed1988

Stability and motion of a hairpin and the corresponding mismatched duplex: a theoretical exploration using molecular mechanics and normal mode analysis of 2D NMR results on d(GCCGCAGC).

The oligomer d(GCCGCAGC) can adopt two different conformations: i) a duplex with two mismatched A.C base pairs and ii) a hairpin with two C.G base pairs and a single stranded loop. We report molecular mechanics, normal mode analysis, and thermodynamic stability calculations for both structures. We show that the energy-minimized structure and harmonic-dynamics results are in complete agreement with the observed NOE spectrum and imino proton exchange data. We conclude that the high stability of the hairpin structure over the duplex at low salt concentration is due to the higher vibrational entropy contribution to the system free energy by the single stranded loop and to the lack of minor groove phosphate/phosphate electrostatic repulsions that tend to destabilize the duplex.

Base Composition↗
PubMed1987

DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix.

Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond.

Adenosine↗
PubMed1987

DNA hairpin structures in solution: 500-MHz two-dimensional 1H NMR studies on d(CGCCGCAGC) and d(CGCCGTAGC).

A hairpin structure contains two conformationally distinct domains: a double-helical stem with Watson-Crick base pairs and a single-stranded loop that connects the two arms of the stem. By extensive 1D and 2D 500-MHz 1H NMR studies in H2O and D2O, it has been demonstrated that the DNA oligomers d(CGCCGCAGC) and d(CGCCGTAGC) form hairpin structures under conditions of low concentration, 0.5 mM in DNA strand, and low salt (20 mM NaCl, pH 7). From examination of the nuclear Overhauser effect (NOE) between base protons H8/H6 and sugar protons H1' and H2'/H2", it was concluded that in d(CGCCGCAGC) and d(CGCCGTAGC) all the nine nucleotides display average (C2'-endo,anti) geometry. The NMR data in conjunction with molecular model building and solvent accessibility studies were used to derive a working model for the hairpins.

DNA↗