Self-complementary Oligodeoxyribonucleotides Research Articles

Self-complementary oligodeoxyribonucleotides incorporating L-related isodeoxynucleosides: synthesis, physical characterization, enzymology, and CD studies.

The synthesis of self-complementary oligodeoxyribonucleotides (14-16, 18) incorporating the L-related isodeoxynucleotides, isodA (1) and/or isodT (2), is described. For this purpose, the fully protected phosphoramidite building blocks 10 and 12 were synthesized. The conceptually new oligomers exhibit high resistance towards exonucleases. Temperature- and concentration-dependent CD spectral data and accompanying thermodynamic parameters of the oligomers show that isodA-isodT or mixed dA-isodT/isodA-dT base pairs participate in duplex stabilization.

Novel oligodeoxyribonucleotides incorporating L-related isodeoxynucleosides: Solid phase synthesis, enzymology, and CD studies

The synthesis of self-complementary oligodeoxyribonucleotides incorporating the L-related isodeoxynucleosides, isodA, (1) and/or isodT (4) is described. These novel oligomers exhibit high resistance towards exonucleases. Temperature- and concentration-dependent CD spectra and thermodynamic data of the oligomers show that isodA-isodT base pairs participate in duplex stabilization.

Self-complementary oligodeoxyribonucleotides containing 2'-O-(anthraquinone-2-methyl)adenosine.

Incorporation of an intercalating agent into an oligodeoxynucleotide (ODN) has the potential to enhance binding affinity upon duplex formation, to increase ODN hydrophobicity, and to enhance resistance to nuclease hydrolysis. Site-specific intercalation has been achieved through the synthesis of 2'-O-(anthraquinone-2-methyl)adenosine(rA*) and its incorporation into the palindromic dodecanucleotide d(CGCrA*CATGTGCG). Melting temperature, CD spectra, 1D and 2D (DQF-COSY and NOESY) NMR spectra, and molecular models were obtained and compared with the unmodified dodecamer. The data clearly establish that intercalation of the anthraquinone ring into a predominantly B-type helix occurs between the A4-T9 and C5-G8 base pairs, significantly stabilizing the duplex and enhancing the hydrophobicity of the ODN.

Synthesis of Base-Modified Oligonucleotides Containing 6- and 7-Aryl Lumazines

Abstract 6-Phenyl, 7-phenyl, 6-(4-biphenyl)-, 7-(4-biphenyl)lumazine N1-(2′-deoxy-D-ribofuranosides) were synthesized and incorporated in the different positions of self-complementary oligodeoxyribonucleotides, and the influence of modifications on the melting points of duplexes was studied.

Effect of DNA base sequence on the configuration of deoxyadenosine adducts formed by the Fjord region diol epoxide, (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

The carcinogen (+)-(1R,2S,3R,4S)-3,4-dihydroxy-1,2-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene (in which the 4-OH group and epoxide oxygen are cis) was reacted with duplexes formed from self-complementary oligodeoxyribonucleotides, producing 1S or 1R configured adducts through trans or cis epoxide ring opening, respectively, by the exocyclic amino group of a central target A. Sequences containing 5'-AT-3' generated much higher S vs R ratios than the average of 3.38 observed with calf thymus DNA samples, while sequences containing 5'-TA-3' generated much lower ratios. Sequences with G in the position immediately 5' to the central AT or TA, and C in the position immediately 3', generated moderately higher ratios than did sequences with adjacent 5'C and 3'G. When thymidine was replaced by deoxyuridine in several sequences, the ratios of S vs R configured dA adducts and dA adducts vs dG adducts were substantially and uniformly reduced, but otherwise varied with the choice of nearest neighbors in patterns similar to those observed with the T containing sequences. Two hypothetical mechanisms are proposed to explain the effect of nearest neighbors on the S vs R dA adduct ratio; in both, diol epoxide intercalation precedes covalent bonding. In one mechanism, intercalation to the 5' side of the target A yields an S adduct while intercalation to the 3' side yields an R adduct, and the extent of adduct formation follows the nearest neighbor series G > C > T.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography of the photoproducts of nucleic acid components: III. Detection of the secondary structure differences in sequence isomeric self-complementary oligonucleotides

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect differences in the secondary structures of two self-complementary oligodeoxyribonucleotides. The [d(CCTTTAAAGG)] 2 duplex assumes an ordinary B-conformation in aqueous solution, while [d(GGAAATTTCC)] 2 is known to contain in its central part a stretch of a more rigid B′-form conformation with significantly lowered fluctuational mobility of base pairs. The latter factor causes a marked difference in the amounts of thymine cyclobutane photodimers formed under UV irradiation of corresponding duplexes as revealed by chromatography of two single-stranded oligonucleotides. Increasing the temperature below the duplex melting temperature (stimulation of the B′-B structural transition) results in an increase in photodimer formation that was inhibited in the B′-form. Thus, we demonstrate the usefulness of RP-HPLC for duplex DNA structural studies.

X-Ray Crystallographic Studies of the A-Form of DNA

Abstract The three-dimensional structure in the crystalline state of the self-complementary oligodeoxyribonucleotides d(GCCCGGGC) and d(GGGATCCC) is described and compared with other DNA fragments in the A-form.

An NMR investigation of the binding of the anticancer drug actinomycin D to oligodeoxyribonucleotides with isolated 5'd(GC)3' binding sites.

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with one GC binding site [d(ATATGCATAT) (1), d-(ATACGCGTAT) (2), and d(ATATACGCGTATAT) (3)] and with two GC sites [d(ATGCATGCAT) (4)]. At R = 1 (molar ratio of ActD to oligomer duplex) ActD caused a doubling of the number of imino proton signals at, and adjacent to, the GC binding site of 1. One of the G.C base pair signals shifted upfield while the other shifted downfield. Both of the signals for the A.T base pairs adjacent to the binding site shifted downfield. All imino proton signals of 2 and the longer sequence, 3, shifted upfield on binding of ActD to the GC site, indicating a sequence-dependent change in base stacking on complex formation. For both 1 and 2 addition of ActD resulted in a similar pattern of three downfield 31P NMR signals. The two most downfield signals have chemical shift and temperature dependence which are characteristic of phosphate groups at isolated intercalation sites. At R = 1 the ActD complex with 4 has very complex spectra with both upfield and downfield A.T and G.C imino signals. All these data were consistent with two 1:1 complexes with the unsymmetrical phenoxazone ring adopting both of the two possible orientations.(ABSTRACT TRUNCATED AT 250 WORDS)

1H and 31P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites.

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.

31P NMR investigation of fourteen self-complementary oligodeoxyribonucleotides (8- mer to 14- mer) treated with platinum complexes: the downfield 31P NMR signal characteristic of anti-cancer drugs is observed only for molecules with adjacent G residues

The reaction products of fourteen self- complementary oligodeoxyribonucleotides (from 8 to 14 bases in length) treated with cis-PtA 2Cl 2 (A= amine) and ten treated with trans-Pt(NH 3) 2 Cl 2 were studied by 31P NMR spectroscopy. Treatments were carried out under high salt (0.1 M NaNO 3) and low temperature (12 °C) conditions which favor the duplex form. A non-transient downfield 31P NMR signal was observed at ca. −3.0±0.1 ppm (relative to trimethyl phosphate) for each cis-Pt(NH 3) 2Cl 2- and Pt(en)Cl 2 -treated oligodeoxyribonucleotide which contained an NGG moiety. In some cases two downfield signals were observed. One downfield signal was attributed to the phosphate group in a platinated GpG moiety for the single-stranded oligomers. The additional downfield signals were attributed to phosphate groups in distorted unplatinated moieties in single-stranded forms. For the Pt(en)Cl 2-treated octadeoxyribonucleotide, d(GGAATTCC) 2, a 31P0 NMR signal was observed at −3.41 ppm. The relatively hight field position of this signal is most likely due to the absence of a nucleotide or phosphate group 5' to the GG moiety. No signals were observed outside the normal shift region for any of the oligodeoxyribonucleotides treated with trans-Pt(NH 3) 2Cl 2. Non-transient downfield 31P signals were not observed for any of the cis-PtA 2Cl 2-treated dodeca- and tetradecadeoxyribonucleotides which did not contain a GG sequence but did contain reactive A, GA, AG and GNG moieties. For the two tetradecadeoxyribonucleotides which contain AG sequences, d(TATAGCTAGCTATA) 2 and d(ATAGCTTAAGCTAT) 2, treatment with Pt(en)Cl 2 produced 31P signals shifted upfield from the main signal by ca. 1.4 ppm. These signals were observed only at low temperature (15 °C). Therefore, for these oligodeoxyribonucleotides at low temperature, Pt(en)Cl 2 induced distortions severe enough to be observed by 31P NMR spectroscopy. This survey provides additional evidence that Pt-crosslinked GG moieties are responsible for the occurrence of the downfield signal observed for DNA treated with anti-cancer active Pt drugs. The evidence is strong that such downfield signals must include the signal from the phosphate group in the platinated GpG moiety. However, it is possible that the signal also contains contributions from other phosphate groups in the vicinity of the platinated GpG moiety.

T4 RNA ligase catalyzed synthesis of base analogue-containing oligodeoxyribonucleotides and a characterization of their thermal stabilities

Self-complementary oligodeoxyribonucleotides containing the base analogues 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, uracil, and 5-bromouracil were synthesized by a general method that allows incorporation of the analogues at specific positions. The method uses chemically synthesized partial sequences but circumvents the need for protected base analogues by incorporating their unprotected 3',5'-bisphosphate derivatives enzymatically. T4 RNA ligase was used to add the analogues to the oligodeoxyribonucleotides with yields from 54 to greater than 95 percent. Oligodeoxyribonucleotides were joined to the oligodeoxyribonucleotides containing the analogues at their 3'-termini in yields from 22 to 81 percent. The high yields obtained in these joinings suggest that RNA ligase should be of general use for the specific incorporation of other deoxyribonucleotide analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.

Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions.

The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of high concentrations of a variety of nonspecific polymers such as polyethylene glycol, Ficoll, bovine plasma albumin, or glycogen. Blunt-end ligation of small self-complementary oligodeoxyribonucleotides is also stimulated. The use of polyethylene glycol 6000 in such systems is characterized in some detail. Conditions are suggested which either greatly increase the rate of formation of and size of linear ligation products or which allow smaller but significant improvements in the amounts of circular ligation products.