Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Abstract

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2′-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

Photochemical modifications for DNA/RNA oligonucleotides

“…7-Ntroindole and 5-nitroindole DNA nucleosides have both been shown to lower the T m of DNA duplexes. 7-Nitroindole was slightly more destabilizing than 5-nitroindole: 82 13-15 C lower T m for 7nitroindole than that for unmodied DNA versus 10-11 C lower T m for 5-nitroindole in the same 11-bp DNA duplex context. 82 Biological applications.…”

“…7-Nitroindole was slightly more destabilizing than 5-nitroindole: 82 13-15 C lower T m for 7nitroindole than that for unmodied DNA versus 10-11 C lower T m for 5-nitroindole in the same 11-bp DNA duplex context. 82 Biological applications. Light-induced photocleavage of nitroindole was used to induce controlled release of DNAbinding proteins such as NF-kB as a part of the catch-andrelease DNA decoys where 7-nitroindole could be used in place of regular dG's in the NF-kB binding sequence.…”

“…6). 82 Subsequent mild basic or thermal treatment leads to cleavage of the DNA backbone via band d-elimination at the abasic site. 82 Reaction characteristics.…”

“…82 Subsequent mild basic or thermal treatment leads to cleavage of the DNA backbone via band d-elimination at the abasic site. 82 Reaction characteristics. The photocleavage reactions of nitroindole-modied DNA nucleoside accompany changes in the absorption spectra.…”

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2'-deoxyadenosine mimic

“…Subsequently, mild alkaline or thermal treatment leads to DNA strand cleavage through a β-elimination reaction ( Figure 2 ). DNA polymerase recognition of d(7-Ni) incorporated either in a DNA matrix or to a primer as its corresponding triphosphate has also been studied ( 23 ). It was shown that the modified base was not specifically recognized by the enzyme.…”