Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

Abstract

Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide.

“…1). Psoralens have previously been attached to oligonucleotides targeted to single-stranded nucleic acid sequences (15)(16)(17)(18)(19). The psoralen ring was either chemically attached to the oligonucleotide via its C-8 (15) or its C-4' (16-18) position or photochemically linked via its furane ring to thymine at a unique 5'-TpA-3' sequence of the oligonucleotide (19).…”

Site-specific and photo-induced alkylation of DNA by a dimethylanthraquinone-oligodeoxynucleotide conjugate

“…Related naphthoquinone conjugates did not exhibit this activity and covalent attachment of a naphthoquinone had no effect on duplex hybridization (12). Interestingly, psoralen intercalates into duplex DNA, but it does not seem to stabilize hybridization when linked as a oligodeoxynucleotide-psoralen conjugate (7,(34)(35)(36). …”

“…However, affinity can usually be regained and metabolic stability further enhanced by appending an intercalator or alkylating agent to a terminus of the oligomer (6). Psoralen has been used widely in this manner, since it can covalently cross-link single-and double-stranded nucleic acids upon exposure to near UV light (7)(8)(9)(10). Our research has focused on a series of related reagents that similarly express an inducible ability to alkylate nucleic acids.…”

Conjugated Oligonucleotides for Biochemical Applications

“…Psoralene is a planar molecule able to intercalate preferentially among TA base pairs to give, after UV irradiation, a reversible [2+2] cycloadduct with the C5-C6 atoms of the thymidine rings on the two facing strands. This molecule had been conjugated to antisense DNA since the beginning of this methodology [46,47]. Our original methodology was based on the efficient C8 alkylation of the adenine ring by a sulfur atom at the end of a bifunctional dithiol alkyl linker (Scheme 6).…”