Synthesis and characterization of 5-[(4-Azidophenacyl)thio]uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analogue.
ABSTRACT The synthesis, isolation, and characterization of a new photo-cross-linking uridine 5'-triphosphate analogue are described. This nucleotide analogue, 5-[(4-azidophenacyl)thio]uridine 5'-triphosphate (5-APAS-UTP), contains an aryl azide group approximately 10 A from the uridine ring. The azide is photoactivated by irradiation at 300 nm, resulting in covalent attachment of the nucleotide to adjacent molecules. The nucleotide can be desulfurated with Raney nickel to cause molecular cleavage between the base and the aryl azide. Desulfuration yields uridine 5'-triphosphate and p-azidoacetophenone. If the analogue is cross-linked to another molecule, desulfuration leaves the analogue's acetophenone group attached to that molecule. This effectively leaves behind a molecular tag on molecules that interact with the uridine analogue either as monomeric nucleotide or as part of an RNA molecule. This nucleotide analogue can be incorporated into internal positions in RNA by transcription in vitro with Escherichia coli RNA polymerase. It can therefore be used to examine interactions between RNA and other molecules (e.g., proteins or nucleic acids). Because the sulfur atom can be selectively removed, the covalent bonds formed between analogue-containing RNA and other molecules can be cleaved, when desired, to facilitate identification of the cross-linked molecules and RNA nucleotides in the cross-linked complex.
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ABSTRACT: A new photocrosslinking purine analog was synthesized and evaluated as a transcription substrate for Escherichia coli RNA polymerase. This analog, 8-[(4-azidophenacyl)thio]adenosine 5'-triphosphate (8-APAS-ATP) contains an aryl azide photocrosslinking group that is attached to the ATP base via a sulfur-linked arm on the 8 position of the purine ring. This position is not involved in the normal Watson-Crick base pairing needed for specific hybridization. Although 8-APAS-ATP could not replace ATP as a substrate for transcription initiation, once stable elongation complexes were formed, 8-APAS-AMP could be site-specifically incorporated into the RNA, and this transcript could be further elongated, placing the photoreactive analog at internal positions in the RNA. Irradiation of transcription elongation complexes in which the RNA contained the analog exclusively at the 3' end of an RNA 22mer, or a 23mer with the analog 1 nt from the 3' end, produced RNA crosslinks to the RNA polymerase subunits that form the RNA 3' end binding site (beta, beta'). Both 8-APAS-AMP and the related 8-azido-AMP were subjected to conformational modeling as nucleoside monophosphates and in DNA-RNA hybrids. Surprisingly, the lowest energy conformation for 8-APAS-AMP was found to be syn, while that of 8-azido-AMP was anti, suggesting that the conformational properties and transcription substrate properties of 8-azido-ATP should be re-evaluated. Although the azide and linker together are larger in 8-APAS-ATP than in 8-N(3)-ATP, the flexibility of the linker itself allows this analog to adopt several different energetically favorable conformations, making it a good substrate for the RNA polymerase.Nucleic Acids Research 06/2000; 28(9):1849-58. · 8.81 Impact Factor
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ABSTRACT: The use of the reaction of azide and alkyne cycloaddition for the synthesis of nucleic acid conjugates and DNA oligomer analogues is considered. The data on chemical and enzymatic techniques of azides and alkynes introduction into DNA are summarized.Bioorganicheskaia khimiia 01/2010; 36(4):437-81.
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ABSTRACT: Labeled nucleic acid probes are used as diagnostic tools by detecting changes in gene expression upon hybridization to target RNAs or DNAs that are related to specific disease genes. 5-[S-(2, 4-Dinitrophenyl)-thio]-2'-deoxyuridine analog represents an excellent nucleic acid label, containing the DNP group which functions both as a probe and as a precursor for the introduction of a variety of fluorescent groups. This study describes thermal denaturation hybridization experiments with oligonucleotides containing the 5-[S-(2,4-dinitrophenyl)-thio]-2'-deoxyuridine analog. Using molecular modeling techniques, the effects of this analog on the hybrid structure and stability were examined, including (i) analog conformation, (ii) hydrogen bonding, (iii) stacking interactions and (iv) hybrid helical geometry. This analog does not prohibitively affect the hybrid thermal stability and incorporation of the analog does not compromise the structural integrity of the double helix. In particular, the sequence-dependence of the analog effects and the dependence on the modification site relative to the end(s) of the helix were investigated. Findings described here should provide guidelines in the rational design of nucleic acid probes.Journal of Molecular Recognition 01/1999; 12(6):337-45. · 3.01 Impact Factor