Mechanism of Site-Specific Psoralen Photoadducts Formation in Triplex DNA Directed by Psoralen-Conjugated Oligonucleotides

Abstract

Triplex-formation oligonucleotides attached with a photoreactive psoralen molecule (psoTFO) can be used to induce site-specific DNA damage and to control gene expression. Inhibition of transcription by psoralen-cross-linked triplexes results in both arrest and termination of RNA Pol II transcriptional complexes during elongation. To understand the relationship between triplex psoralen cross-linking products and the fate of RNA Pol II elongation complexes, it is important to delineate the mechanism for creating site-specific psoralen photoadducts in a target duplex DNA. To investigate the mechanism of photoadduct-formation by psoralen photo-cross-linking, triplex structures were generated by targeting a DNA duplex with psoTFOs of different lengths. The psoralen photoadducts were then analyzed after UV irradiation, which initiates the psoralen cross-linking reaction. Our results demonstrated that UV irradiation of triplexes formed between a psoTFO and a DNA duplex generated two distinct groups of psoralen photoadducts: monoadducts and psoralen interstrand cross-link products. The formation of these psoralen photoadducts was also photoreversible through exposure to short wavelength UV irradiation. The length of a psoTFO was shown to establish the position at which psoralen was added to the target DNA duplex and determined which photoadducts products formed predominantly. Kinetic experiments that monitored the formation of the psoralen photoadducts also suggested that the length of the psoTFO influenced which photoadducts were preferentially formed at faster rates. Taken together, these studies provide new insight into the mechanism associated with the formation of psoralen photoadducts that are directed by psoTFO during triplex formation.

Quantitative Analysis of DNA Interstrand Cross-Links and Monoadducts Formed in Human Cells Induced by Psoralens and UVA Irradiation

“…ODNs containing psoralen mono-adducts were prepared by photo-reversal reaction of ICL-containing ODNs 22. The 365-nm irradiation mixture, which contained both ICL and unreacted starting material, was immediately irradiated on ice with short-wavelength UV light centered at 254 nm (Spectronics Corp., Westbury, NY).…”

Contribution of Biotechnological Tools in the Enhancement of Secondary Metabolites in Selected Medicinal Climbers

“…Photoreactive psoralen molecule forms complex with oligonucleotides. This complex incites site-specifi c DNA damage and regulates gene expression (Ping and Pana 2005 ). Seed extract of P. corylifolia inhibits tumour growth and stimulates natural killer activity (Latha et al 2000 ) .…”

Furanocoumarins

“…The DNA itself can be modified by compounds with reactive groups, such as epoxides. Interaction between furanocoumarin and DNA occurs in two steps: (a) intercalation of coumarin between two base pairs of the DNA and (b) covalent photoconjugation through 3,4 and 2 0 ,3 0 double bonds of bifunctional furanocoumarins [100]. Furanocoumarins such as angelicin, which only form monoadducts (involving the 3,4 double bond), induce mutation at a frequency comparable to ultraviolet light of 254 nm [101].…”