Biomedical subjects

N S Kondo

Publications and source records attributed to N S Kondo.

16 recordsLinked to original sources

PubMed2000

Triplex formation by psoralen-conjugated chimeric oligonucleoside methylphosphonates.

Interactions between nuclease-resistant, 5'-psoralen-conjugated, chimeric methylphosphonate oligodeoxyribo- or oligo-2'-O-methylribo-triplex-forming oligomers (TFOs) and a purine tract found in the envelope gene of HIV proviral DNA (env-DNA) were investigated by gel mobility shift assays or by photo-cross-linking experiments. These chimeric TFOs contain mixtures of methylphosphonate and phosphodiester internucleotide bonds. A pyrimidine chimeric TFO composed of thymidine and 5-methyl-2'-deoxycytidine (C), d-PS-TpCpTpCpTpCpTpTpTpTpTpTpCpTpC (1mp) where PS is trimethylpsoralen and p is methylphosphonate, forms a stable triplex with env-DNA whose dissociation constant is 1. 3 microM at 22 degrees C and pH 7.0. The dissociation constant of chimeric TFO 2mp, d-PS-UpCpTpCpTpCpTpUpTpUpTpUpCpTpC, decreased to 400 nM when four of the thymidines in 1mp were replaced by 5-propynyl-2'-deoxyuridines (U), a result consistent with the increased stacking interactions and hydrophobic nature of 5-propynyl-U. An even greater decrease, 470 -50 nM, was observed for the all-phosphodiester versions of 1mp and 2mp. The differences in behavior of the chimeric versus the all-phosphodiester oligomers may be related to differences in the conformations between the propynyl-U-substituted versus the nonsubstituted TFOs. Thus, in the chimeric oligomer, the stabilizing effect of the propynyl-U's may be offset by the reduced ability of the methylphosphonate backbone to assume an A-type conformation, a conformation that appears to be preferred by propynyl-U-containing TFOs. A chimeric oligo-2'-O-methylribopyrimidine with the same sequence as 1mp also formed a stable triplex, K(d) = 1.4 microM, with env-DNA. In contrast to the behavior of the pyrimidine TFOs, antiparallel A/G oligomers and parallel or antiparallel T/G oligomers did not form triplexes with env-DNA, even at oligomer concentrations of 10 microM. This lack of binding may be a consequence of the low G content (33%) of the triplex binding site. Irradiation of triplexes formed between the pyrimidine TFOs and env-DNA resulted in formation of photoadducts with either the upper-strand C or the lower-strand T at the 5'-CpA-3' duplex/triplex junction. No interstrand cross-links were observed. The presence of a 5-propynyl-U at the 5'-end of the oligomer caused a reduction in the amount of upper-strand photoadduct but had no effect on photoadduct formation with the lower strand, suggesting that increased stacking interactions caused by the presence of the 5-propynyl-U change the orientation of psoralen with respect to the upper-strand C. The ability of chimeric methylphosphonate TFOs to bind to DNA, combined with their resistance to degradation by serum 3'-exonucleases, suggests that they may have utility in biological experiments.

Base Sequence↗

PubMed2000

Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages.

Preliminary investigations of the physical properties of oligonucleotide analogs that contain alternating methylphosphonate/phosphodiester linkages are described. An alternating oligo-2'-O-methylribonucleoside methylphosphonate, oligomer 1676, whose sequence is complementary to the upper hairpin region of human immunodeficiency virus TAR RNA, has been synthesized. This 15-mer forms a very stable duplex with its complementary RNA target, whose melting temperature is 71 degrees C. Introduction of two mismatched bases reduces the melting temperature by 16 degrees C. Similar results were obtained with the all-phosphodiester version of oligomer 1676, which demonstrates that introduction of the methylphosphonate linkages does not significantly perturb the ability of the oligo-2'-O-methylribonucleoside methylphosphonate to bind to RNA. Unlike the phosphodiester oligomer, however, oligomer 1676 is completely resistant to hydrolysis by the 3'-exonuclease activity found in mammalian serum. The interactions between nuclease-resistant, 5'-psoralen-derivatized, alternating oligo-2'-deoxypyrimidine methylphosphonates and double-stranded DNA were also studied. A 15-mer that contains thymine, 5-methylcytosine, and 5-propynyl-uracil forms a triplex with a polypurine tract found in the env gene of human immunodeficiency virus proviral DNA with an apparent dissociation constant of 400 nM at 22 degrees C. Maximal triplex formation by these oligomers is observed at approximately 2.5 mM magnesium, whereas maximal triplex formation by the corresponding all-phosphodiester oligomers occurs between 10 and 20 mM magnesium. This reduced magnesium dependence most likely results from reduced charge repulsion between the backbones of the methylphosphonate oligomer and purine strand of the target. The nuclease stability and ability of the methylphosphonate oligomers to form stable complexes with their target nucleic acids suggest that these oligomers are potential candidates for use as antisense or antigene agents in cell culture.

Binding Sites↗

PubMed1984

X-ray structure of cytidine-5'-O-dimethylphosphate. Novel stacking between the ribosyl O(2') hydroxyl oxygen atom and the base.

The anionic oxygen atoms of the phosphodiester backbone of RNA and DNA are particularly susceptible to esterification by many mutagenic and carcinogenic alkylating agents. To better understand the geometric, electronic and conformational properties of the alkylated sugar phosphate moiety, the X-ray structure of the phosphotriesterified nucleotide, cytidine-5'-O-dimethylphosphate (C11H18N3O8P), was undertaken. The compound crystallizes in the monoclinic space group P2, with unit cell parameters of a = 5.741(2), b = 11.625(1), c = 11.425(1)A, beta = 94.43(2) degrees. The structure was solved by direct methods and refined by block-diagonal least-squares technique to an R index of 0.034 (Rw = 0.046). The D-ribofuranosyl ring is in the 3T2 twist conformation (P = 13.1(2) degrees, tau m = 36.7(2) degrees) and the conformation about the C(1')-N(1) glycosyl bond is anti (XCN = 8.3(2) degrees). The four P-O bond lengths are significantly shorter than those of the nonalkylated nucleotides. The three sets of phosphodiester linkages, (omega 'A, omega A), (omega 'B, omega B) and (omega 'C, omega C), take the (g-,t), (t,g) and (g-,t) conformations, respectively. There is no base-base or alkyl-base stacking, however, a novel intermolecular stacking is found between the ribosyl O(2') hydroxyl oxygen atom and a neighboring pyrimidine ring. This hydroxyl-base stacking interaction may have implications in the stabilization of the tertiary and quarternary structure of ribonucleic acids and nucleic acid-protein complexes.

Chemical Phenomena↗

PubMed1977

Conformational properties of purine-pyrimidine and pyrimidine-purine dinucleoside monophosphates.

The detailed conformational features and dynamics of heterodinucleoside monophosphates ApU, ApC, GpU, GpC, UpA, CpA, UpG, and CpG have been studied in aqueous solution by high field nuclear magnetic resonance (NMR) spectroscopy. Analysis of the resultant NMR parameters leads to a number of discernible trends throughout the series. Thus the ribose rings of the dimers exist as equilibrium mixtures of C(2')-endo(2E) in equilibrium C(3')-endo(3E) conformers with a proclivity for the 3E pucker in most cases; the C(4')-C(5') bonds of both nucleotidyl units show significant preference (74-96%) for a gg conformation and the dominant conformer (85-89%) about C(5')-O(5') is g'g'. Orientation about the C(3')-O(3') bond is coupled to the ribose conformational equilibrium and the system exists with a bias for the 3Eg- coupled conformation in which the H(3')-C(3')-O(3')-P dihedral angle occupies the narrow range of 33-35 degrees. Dimerization, on the average, causes about 10% increase in gg and g'g' populations and the g-domain becomes increasingly populated about the C(3')-O(3') bond. The ribose equilibrium 2E in equilibrium 3E shifts in favor of 3E upon dimerization, the effect being very conspicuous for the pu-py series (similar to 40 yields 60%) and less noticeable for the py-pu systems (similar to 47 yields 58%), clearly suggesting a correlation between sequence and ribose conformational equilibrium. The temperature and dimerization data for the heterodinucleoside monophosphates show that the transition 2E yields 3E is directly related to XCN changes induced by dimerization and stacking. Analysis of the ribose coupling data shows that the percentage populations of stacked species vary from dimer to dimer with GpC displaying a maximum of 45% stacked population and UpG about 10%. However, in general, the pu-py dimers show a higher preference (27-45%) for stacked conformations than py-pu dimers (10-25%). It is proposed that the pronounced deshielding of H(5') of the 5'-nucleotidyl units upon dimerization is associated with the presence of right-handed stacks (g-g-), whereas the chemical shift trends of H(5') and H(5') of 3'-nucleotidyl units are due to the presence of left-handed stacks (g+g+) in all the dimers. In pu-py dimers, the population of the g-g- species is found to be greater than that of g+g+. Also the population of g-g- stacks in pu-py dimers is generally greater than in their corresponding matched py-pu dimers. Thus the base sequence has not only an explicit effect on the overall populations of stacked species, but also on the handedness of the stacks. The present results further confirm the interdependence of conformational bonds throughout the nucleotidyl framework.

Deuterium↗

PubMed1976

Conformational properties of dinucleoside monophosphates in solution: dipurines and dipyrimidines.

In order to obtain information about the conformational features in a polyribonucleotide at the nearest neighbor level, detailed nuclear magnetic resonance studies of the dinucleoside monophosphates ApA, ApG, GpA, UpU, CpC, UpC, and CpU were undertaken. Proton spectra were recorded at 100, 220, 270, or 300 MHz for D2O solutions, 0.01-0.03 M, pD 7.4 at 20+/-2 degrees C. Spectra of ApA, ApG, UpU, and UpC were also recorded in the temperature range of 70-90 degrees C. Unambiguous signal assignments of all proton resonances were made with the aid of selectively deuterated dimers. Complete, accurate sets of nuclear magnetic resonance (NMR) parameters were derived for each nucleotidyl unit by simulation-iteration methods. A complete set of chemical shift and coupling constant data was also obtained for all the constituent monomeric units at a concentration and ionization state comparable to that of the dimers. Conformational properties were evaluated quantitatively for most of the bonds in the dinucleoside monophosphates using procedures developed in earlier studies. All of the dimers have a flexible conformational framework in aqueous solution. While flexibility is allowed and alternate conformations are accessible, these molecules nevertheless attempt to achieve conformational identity by showing preferences--sometimes overwhelming preferences--for certain orientations. Thus the ribose rings exist as equilibrium mixtures of C2'-endo in equilibrium C3'-endo conformers with a bias for the C3'-endo pucker in most cases. The C4'-C5' bonds of both nucleotidyl units show significant preference (70-85%) for a gg conformation. Similarly, the dominant conformer (80-90%) about C5'-O5' is g'g'. Even though an unambiguous determination of the orientation about C3'-O3' cannot be made, there is suggestive evidence that the orientation of the 3' phosphate group is coupled to the ribose conformational equilibrium and it is likely that a 3Eg- in equilibrium 2Eg+ equilibrium exists with a bias for the 3Eg- coupled conformation in which the H3'-C3'-O3'-P dihedral angle is about 34-38 degrees. The individual nucleotidyl units in the dimers differ in several key ways from corresponding monomer conformations. Specifically, the ribose equilibrium C2'-endo in equilibrium C3'-endo shifts in favor of C3'-endo upon dimerization, the only exception being UpU. The C4'-C5' and C5'-O5' bonding network in the dimer forms a stable conformational unit and no correlation exists in the dimers between the conformational preference of this fragment and ribose conformer population. The temperature data for the dimers and dimerization data clearly indicate that the transition C2'-endo leads to C3'-endo is directly related to XCN changes brought about by dimerization and stacking...

Magnetic Resonance Spectroscopy↗

PubMed1976

Conformational properties of adenylyl-3' leads to 5'-adenosine in aqueous solution.

A detailed 220-MHz NMR study has been made of the conformational properties for the homodinucleotide adenylyl-3' leads to 5'-adenosine, ApA, in D2O. Unambiguous signal assignments of all proton signals were made with the aid of selectively deuterated nucleotidyl units, ApA, ApA, and D-8ApA, and complete, accurate sets of NMR parameters were derived by simulation-iteration methods. Sets of limiting chemical shifts and coupling values were also obtained for ApA and constituent monomers 3'-AMP and 5'-AMP at infinite dilution and at identical ionization states for assessment of dimerization effects. Conformational properties were evaluated quantitatively for most of the conformational bonds of ApA and these are consistent with two compact folded dynamically averaged structures, a base-stacked right helical structure, I, characterized as anti, C3'-endo, g-, w,w' (320,330 degrees), g'g', gg, C3'-endo, anti, and a more loosely base-stacked loop structure, II, with anti, C3'-endo, g-, w,w' (80 degrees, 50 degrees), g'g', gg, C3'-endo, anti orientations. Dimerization produces a number of nucleotidyl conformational changes including a shift in ribose equilibrium C2'-endo (S) in equilibrium C3'-endo (N) in favor of C3'-endo in both Ap- and -pA (60:40 vs. 35:65 in monomers), a change in glycosidic torsion angle chiCN toward 0 degrees, and a greater locking-in of rotamers along bonds involved in the phosphodiester backbone. Moreover, there is clear evidence that the transitions from S leads to N forms and chiCN leads to 0 degrees are directly related to base stacking in ApA. Finally, ApA exists in solution as an equilibrium between I, II and an unstacked form(s) with as yet undetermined conformational features. Since C4'-C5', C5'-O5', and C3'-O3' bonds possess exceptional conformational stabilities, it is proposed that destacking occurs primarily by rotation about P-O5' and/or O3'-P. Predominant factors influencing the overall ApA conformation are thus base-base interaction and flexibility about P-O5' and O3'-P, with change of ribose conformation occurring in consequence of an alteration of chiCN, the latter in turn being governed by the need for maximum eta overlap of stacked adenine rings.

Adenine Nucleotides↗

PubMed1976

The effect of (2'-5') and (3'-5') phosphodiester linkages on conformational and stacking properties of cytidylyl-cytidine in aqueous solution.

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.

Cytosine↗

PubMed1975