Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA (original) (raw)
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Nucleic Acids Research, 1991
Chemical ligation of oligonucleotides in doublestranded helices has been considered in its structuralkinetic aspect. A study was made of (i) two series of DNA duplexes with various arrangements of reacting groups in the ligation junction induced by mispairing or by alteration of furanose structure (the replacement of dT unit with rU, all, IU, xU, dxT ones) and of (ii) eight synthetic water-soluble carbodiimides with different substituents at N1 and N3 atoms. We assumed that some information on the local structure of modified sites in the duplex can be obtained from kinetic parameters of oligonucleotide coupling reaction. The ratio of kinetic constants k 3 /(k 2 + k 3) for productive and nonproductive decomposition of the activated phosphomonoester derivative apparently reflects the reaction site structure: for a given duplex this parameter is virtually independent of the condensing agent composition. Based on the analysis of the chemical ligation kinetics a suggestion has been made about the conformation of some modified units in the double helix.
Formation of phosphonester bonds catalyzed by DNA polymerase
Nucleic Acids Research, 1992
3'-Fluoro-2',3'-dideoxythymidine 5'-(a-methylphosphonyl)-3,Bdiphosphate and 2'-deoxythymidine-5'-(a-methylphosphonyl)-f,ydiphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase a from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E.co1i DNA polymerase 1, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases a and e from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned.
Biochemistry, 1996
We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3′-3′-(RT)-5′-5′-CGC) 2 , in which an R-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3′-3′ and 5′-5′ phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC) 2 . Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the R duplex. Thermodynamic parameters extracted from UV melting experiments show that the R duplex is only slightly less stable than the control. A near complete set of 1 H and 31 P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one-and two-dimensional approaches. Several lines of evidence, in particular, imino 1 H, 31 P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the R and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the R-nucleotide and the unique backbone linkages. Specifically, the R duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the R-nucleotide and the neighboring (3′) cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in RT7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of R sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.
International Journal of Biological Macromolecules, 2001
Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5%-(purine) m (pyrimidine) m -3%. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine-pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson-Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3%-3% phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P= phosphate and X= 1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.
Kinetics and thermodynamics of i-DNA formation: phosphodiester versus modified oligodeoxynucleotides
Nucleic Acids Research, 1998
At slightly acidic or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines have been shown to form a tetrameric structure in which two parallel-stranded duplexes have their hemiprotonated C.C + base pairs face to face and fully intercalated, in a so-called i-motif. Cytosine-rich pyrimidine oligodeoxynucleotides can form an intramolecular i-motif. We have studied the ability of several DNA analogs to fold into this structure. Evidence for folding was provided by thermal denaturation. We have shown that phosphorothioate and phosphodiester oligodeoxynucleotides, but not methylphosphonate or PNA oligomers, may form the i-motif. Four different PS oligodeoxynucleotides were compared with their PO counterparts. In all cases, the melting temperature (T m ) of the phosphorothioate oligomer was equal or slightly inferior (by 2-3_C) to the T m of the natural oligodeoxynucleotide. For long oligodeoxynucleotides, a small change of pH leads to a completely different melting profile: the curves are reversible at pH 6.4 or lower, and a hysteresis is obtained at pH 6.8 or higher; cooling and heating curves were not superimposed, allowing us to determine the rate constants of association (k on ) and dissociation (k off ) as a function of the temperature: these rate constants give linear Arrhenius plots, in agreement with the prediction of the two-state model of association-dissociation. The activation energy E on is strongly negative and, at neutral pH, the phosphorothioate associates and dissociates nine times faster than the phosphodiester oligodeoxynucleotide of identical sequence.
Site-specific inter-strand cross-links of DNA duplexes
Chemical science (Royal Society of Chemistry : 2010), 2013
We report the development of technology that allows inter-strand coupling across various positions within one turn of DNA. Four 2'-modified nucleotides were synthesized as protected phosphoramidites and incorporated into DNA oligonucleotides. The modified nucleotides contain either 5-atom or 16-atom linker components, with either amine or carboxylic acid functional groups at their termini, forming 10 or 32 atom (11 or 33 bond) linkages. Chemical coupling of the amine and carboxylate groups in designed strands resulted in the formation of an amide bond. Coupling efficiency as a function of trajectory distance between the individual linker components was examined. For those nucleotides capable of forming inter-strand cross-links (ICLs), coupling yields were found to depend on temperature, distance, and linker length, enabling several approaches that can control regioselective linkage. In the most favorable cases, the coupling yields are quantitative. Spectroscopic measurements of ...
Impact of Pyrophosphate and O -Ethyl-Substituted Pyrophosphate Groups on DNA Structure
The Journal of Physical Chemistry B, 2007
Design of the novel DNA probes to inhibit specific repair pathways is important for basic science applications and for use as therapeutic agents. As shown previously, single pyrophosphate (PP) and O-ethyl-substituted pyrophosphate (SPP) modifications can inhibit the DNA glycosylase activities on damaged DNA. To understand structural basis of this inhibition, the influence of the PP and SPP internucleotide groups on the helical parameters and geometry of a double-stranded DNA was studied by using molecular modeling tools including molecular dynamics and quantum mechanical-molecular mechanical (QM/MM) approaches. Native and locally modified PP-and SPP-containing DNA duplexes of dodecanucleotide d(C1G2C3G4A5A6T7T8C9G10C11G12) were simulated in aqueous solution. The energies and forces were computed by using the PBE0/6-31+G** approach in the QM part and the AMBER force field parameters in the MM part. Analysis of the local base-pair helical parameters, internucleotide distances and overall global structure at the located stationary points revealed a close similarity of the initial and modified duplexes, with only torsion angles of the main chain being altered in the vicinity of introduced chemical modification. Results show that the PP and SPP groups are built into a helix structure without elongation of the internucleotide distance due to flipping-out of phosphate group from the sugar-phosphate backbone. The mechanism of such embedding has only a minor impact on the base pairs stacking and Watson-Crick interactions. Biochemical studies revealed that the PP and SPP groups immediately 5', but not 3', to the 8-oxoguanosine (8oxodG) inhibit translesion synthesis by a DNA polymerase in vitro. These results suggest that subtle perturbations of the DNA backbone conformation influence processing of base lesions.
Thermodynamic and conformational properties of DNA triplexes containing 3′,3′-phosphodiester bond
Thermochimica Acta, 2001
The thermodynamic characterization of the stability of two DNA triple helices of alternate sequence was obtained by differential scanning calorimetry (DSC). The conformational properties of these triple helices were investigated by circular dichroism (CD) and molecular mechanics. The triplexes under investigation form by way of major groove Hoogsteen association of a Watson±Crick 16-mer duplex with an all pyrimidine 16-mer or 15-mer third strand. The target duplex is composed of two adjacent oligopurine±oligopyrimidine domains where oligopurine sequences alternate on the two duplex strands. Both the third strands contain a 3 H ,3 H -phosphodiester junction, which introduces the appropriate inversion of polarity and let the switch from one oligopurine strand of the duplex to the other. The two-third strands differ for the lack of a cytidine monophosphate in the junction region. Thermal denaturation pro®les indicate the initial loss of the third strand followed by the dissociation of the target duplex with increasing temperature. Transition enthalpies, entropies and free energies were derived from DSC measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region. The thermodynamic data were discussed in relation to structural models. #
Spectroscopic and Calorimetric Characterizations of DNA Duplexes Containing 2-Aminopurine †
Biochemistry, 1996
The base analog 2-aminopurine (AP) strongly promotes A‚T to G‚C and G‚C to A‚T transitions in bacteria and bacteriophage. During DNA replication, the primary mutagenic event involves formation of a heteroduplex with an AP‚C site at a much higher frequency than formation of the corresponding heteroduplex with an A‚C site. It is not known if AP-induced mutagenesis correlates with differences in the thermodynamic properties of an AP‚C versus an A‚C site, or whether interactions involving DNA polymerases are controlling. To address this specific question, and more generally to characterize APcontaining duplexes, we have used a combination of spectroscopic and calorimetric techniques to determine the thermodynamic properties of six 11-mer duplexes. The sequences of these duplexes are identical except for the identity of the variable central base pair which can be either A‚T, A‚C, AP‚T, AP‚C, AP‚A, or AP‚G, and which we use to designate each duplex. Analyses and interpretation of the optically and calorimetrically derived thermal and thermodynamic data on these six duplexes reveal the relative stabilizing influence of the central base pairs to be A‚T > AP‚T > AP‚C > AP‚A > AP‚G > A‚C, with the AP‚C-containing duplex being significantly more stable than the A‚C-containing duplex. In the aggregate, our results suggest that during incorporation, base pair discrimination by DNA polymerases is influenced, in part, by differences in the thermodynamic stabilities of the newly formed base pairs. Abstract published in AdVance ACS Abstracts, September 15, 1996. 1 Abbreviations: AP, 2-aminopurine; NMR, nuclear magnetic resonance spectroscopy; bp, base pair(s). FIGURE 1: Proposed pathway for DNA polymerase-mediated A‚T T G‚C transitions caused by incorporation of AP. Each drawing represents a portion of the DNA duplex after a successive round of replication.