Triplex Formation by a Psoralen-Conjugated Oligodeoxyribonucleotide Containing the Base Analog 8-Oxo-Adenine

Abstract

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.

Recognition of triple-helical DNA structures by transposon Tn7

“…To generate transposition targets containing triplex DNA, several intermolecular triplex targets were constructed by annealing short (13-15 bp) TFOs to plasmids ranging in size from 2.5 to 5.2 kb. To increase stability of these pyrimidine-motif (i.e., Y⅐R⅐Y) triplexes under in vitro transposition reaction conditions (14), TFOs contained nucleoside analogs 5-methylcytosine (19,20) and 8-oxoadenine (21) to negate the low pH requirement of Hoogsteen hydrogen bond formation (22,23), and they also had a psoralen intercalator (24) to allow covalent attachment to the plasmid by photoactivated crosslinking. Once annealed and crosslinked, nearly 80% of the duplex plasmids contained a TFO attached by mono adduct.…”

Triplexes with 8‐Aza‐2′‐Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

“…As regular nucleobases are virtually nonfluorescent, fluorescence has to be introduced into the third strand. Various protocols were developed to probe triplex melting by fluorescence: 1) fluorescent dyes (pyrene) were linked to the triplex, [26,35] 2) the third strand of the 5'-terminus of an oligonucleotide was not labelled with a fluorescent dye but with a quencher, while the purine-rich strand contained a fluorescent dye nearby the quencher, [37] 3) a fluorescence dye was used in intercalator displacement experiments, [38] and 4) base surrogates such as a fluorescent 2'-deoxyuridine with an annealed naphthalene moiety were employed, [39] or the fluorescent 2-aminopurine 2'-deoxyribonucleoside was used instead of 2'-deoxyadenosine within the antiparallel dA-dT-dA triplex motif. [40] To the best of our knowledge, no fluorescent analogue of protonated dC was employed within the dCH + -dG-dC triad.…”

Bioconjugation of Oligonucleotides for Treating Liver Fibrosis

“…Other analogues for substitution of cytosine include 8-oxoadenine (326), pseudoisocytidine (327), and a 6-keto derivative of 5-methylcytydine (328) and 2-aminopyridine (329,330). However, much work still needs to be done on nucleobase analogues regarding their biological applications.…”