Abstract
We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments.
Full text
PDF













Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Chu B. C., Orgel L. E. Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds. Nucleic Acids Res. 1988 May 11;16(9):3671–3691. doi: 10.1093/nar/16.9.3671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cimino G. D., Gamper H. B., Isaacs S. T., Hearst J. E. Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry. Annu Rev Biochem. 1985;54:1151–1193. doi: 10.1146/annurev.bi.54.070185.005443. [DOI] [PubMed] [Google Scholar]
- Cimino G. D., Shi Y. B., Hearst J. E. Wavelength dependence for the photoreversal of a psoralen-DNA cross-link. Biochemistry. 1986 May 20;25(10):3013–3020. doi: 10.1021/bi00358a042. [DOI] [PubMed] [Google Scholar]
- Cole R. S. Light-induced cross-linking of DNA in the presence of a furocoumarin (psoralen). Studies with phage lambda, Escherichia coli, and mouse leukemia cells. Biochim Biophys Acta. 1970 Sep 17;217(1):30–39. doi: 10.1016/0005-2787(70)90119-x. [DOI] [PubMed] [Google Scholar]
- Dall'Acqua F., Marciani S., Rodighiero G. Inter-strand cross-linkages occurring in the photoreaction between psoralen and DNA. FEBS Lett. 1970 Jul 29;9(2):121–123. doi: 10.1016/0014-5793(70)80330-1. [DOI] [PubMed] [Google Scholar]
- Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ericson G., Wollenzien P. Use of reverse transcription to determine the exact locations of psoralen photochemical crosslinks in RNA. Anal Biochem. 1988 Oct;174(1):215–223. doi: 10.1016/0003-2697(88)90538-6. [DOI] [PubMed] [Google Scholar]
- Esposito F., Brankamp R. G., Sinden R. R. DNA sequence specificity of 4,5',8-trimethylpsoralen cross-linking. Effect of neighboring bases on cross-linking the 5'-TA dinucleotide. J Biol Chem. 1988 Aug 15;263(23):11466–11472. [PubMed] [Google Scholar]
- Garrett-Wheeler E., Lockard R. E., Kumar A. Mapping of psoralen cross-linked nucleotides in RNA. Nucleic Acids Res. 1984 Apr 11;12(7):3405–3423. doi: 10.1093/nar/12.7.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Isaacs S. T., Shen C. K., Hearst J. E., Rapoport H. Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA. Biochemistry. 1977 Mar 22;16(6):1058–1064. doi: 10.1021/bi00625a005. [DOI] [PubMed] [Google Scholar]
- Lee B. L., Blake K. R., Miller P. S. Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with synthetic DNA containing a promoter for T7 RNA polymerase. Nucleic Acids Res. 1988 Nov 25;16(22):10681–10697. doi: 10.1093/nar/16.22.10681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee B. L., Murakami A., Blake K. R., Lin S. B., Miller P. S. Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA. Biochemistry. 1988 May 3;27(9):3197–3203. doi: 10.1021/bi00409a011. [DOI] [PubMed] [Google Scholar]
- O'Brien E. J., MacEwan A. W. Molecular and crystal structure of the polynucleotide complex: polyinosinic acid plus polydeoxycytidylic acid. J Mol Biol. 1970 Mar 14;48(2):243–261. doi: 10.1016/0022-2836(70)90159-2. [DOI] [PubMed] [Google Scholar]
- Pieles U., Englisch U. Psoralen covalently linked to oligodeoxyribonucleotides: synthesis, sequence specific recognition of DNA and photo-cross-linking to pyrimidine residues of DNA. Nucleic Acids Res. 1989 Jan 11;17(1):285–299. doi: 10.1093/nar/17.1.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Saffran W. A., Goldenberg M., Cantor C. R. Site-directed psoralen crosslinking of DNA. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4594–4598. doi: 10.1073/pnas.79.15.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shi Y. B., Gamper H., Hearst J. E. Interaction of T7 RNA polymerase with DNA in an elongation complex arrested at a specific psoralen adduct site. J Biol Chem. 1988 Jan 5;263(1):527–534. [PubMed] [Google Scholar]
- Shi Y. B., Gamper H., Hearst J. E. The effects of covalent additions of a psoralen on transcription by E. coli RNA polymerase. Nucleic Acids Res. 1987 Sep 11;15(17):6843–6854. doi: 10.1093/nar/15.17.6843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shi Y. B., Gamper H., Van Houten B., Hearst J. E. Interaction of Escherichia coli RNA polymerase with DNA in an elongation complex arrested at a specific psoralen crosslink site. J Mol Biol. 1988 Jan 20;199(2):277–293. doi: 10.1016/0022-2836(88)90314-2. [DOI] [PubMed] [Google Scholar]
- Shi Y. B., Griffith J., Gamper H., Hearst J. E. Evidence for structural deformation of the DNA helix by a psoralen diadduct but not by a monoadduct. Nucleic Acids Res. 1988 Sep 26;16(18):8945–8952. doi: 10.1093/nar/16.18.8945. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shi Y., Hearst J. E. Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen. Biochemistry. 1986 Oct 7;25(20):5895–5902. doi: 10.1021/bi00368a009. [DOI] [PubMed] [Google Scholar]
- Van Houten B., Gamper H., Hearst J. E., Sancar A. Analysis of sequential steps of nucleotide excision repair in Escherichia coli using synthetic substrates containing single psoralen adducts. J Biol Chem. 1988 Nov 15;263(32):16553–16560. [PubMed] [Google Scholar]
- Van Houten B., Gamper H., Hearst J. E., Sancar A. Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates. J Biol Chem. 1986 Oct 25;261(30):14135–14141. [PubMed] [Google Scholar]
- Wollenzien P. L., Goswami P., Teare J., Szeberenyi J., Goldenberg C. J. The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction. Nucleic Acids Res. 1987 Nov 25;15(22):9279–9298. doi: 10.1093/nar/15.22.9279. [DOI] [PMC free article] [PubMed] [Google Scholar]