::Affinity Photoprobes, LLC::

by Ashok Chavan Ph.D.

Understanding nucleic acid/protein interactions at the molecular level provides insights into many cellular processes.  Photoactive DNA/RNA are powerful tools for such studies⁴.  Photoactive DNA can be synthesized by replacing dTTP with 5N₃-dUTP^5-9.  The protocol for enzymatic sythesis of DNA currently being followed in many laboratories can be utilized with a few minor changes to optimize the system.  It is recommended that DTT be replaced by a monothiol reducing agent such as b-mercaptoethanol⁶.  A radioactive label can then be attached by using [a³²P]dATP, [a³²P]dGTP or [a³²P]dCTP^5-9.   The 8N₃-dATP and 8N₃-dGTP photoprobes may also be used to produce photoactive DNA^8,14,15.  However, they are usually not as readily incorporated into DNA, by most polymerases, as well as 5N₃-dUTP.

Photoactive RNA can similarly be synthesized by replacing UTP with 5N₃-UTP^10-13.   The radioactive label can be attached by using [a³²P]ATP, [a³²P]GTP or [a³²P]CTP.   Additionally 8N₃-ATP and 8N₃-GTP can be used to produce photoactive RNA^16-18.

Advantages of azide substituted (photoactive) DNA/RNA

	The rapidly generated short-lived, reactive nitrene intermediate eliminates long exposure to UV light can damage proteins.  This is a current short coming with direct photo-crosslinking protocols.  In comparison, 5N3-dUTP has a UV sensitivity 7000 times greater that dTTP and over 1000 times greater than dBrUTP5.
	The reactive nitrene intermediate is not generated until photoactivated by UV light, allowing enzymatic studies to be conducted without the loss of the azide group.  The sample can be subsequently irradiated to form the covalent linkage.
	As 5N3-dUTP substitutes well for dTTP, modification to the pyrimidine ring does not perturb the DNA structure, resulting in specificity of interaction.  The N3 group is located within the major groove where the 5CH3 group of Thymidine would normally be present.

Photoprobes for Synthesizing Photoactive DNA

8-Azidoadenosine 2'-deoxytriphosphate

8N3-dATP

	8-Azidoguanosine 2'-deoxytriphosphate	8N3-dGTP
	5-Azidouridine 2'-deoxytriphosphate	5N3-dUTP

Photoprobes for Synthesizing Photoactive RNA

	2-Azidoadenosine 5'-triphosphate	2N3-ATP
	8-Azidoadenosine 5'-triphosphate	8N3-ATP
	8-Azidoguanosine 5'-triphosphate	8N3-GTP
	5-Azidouridine 5'-triphosphate	5N3-UTP

References

⁴Nucleic acid-incorporated azidonucleotides: probes for studying the interaction of RNA or DNA with proteins and other nucleic acids. Sylvers, L.A. an Wower, J. (1993). Bioconjugate Chemistry 4:411-418. (8305509)

⁵5-Azido-2'-deoxyuridine 5'-triphosphate: a photoaffinity-labeling reagent and tool for the enzymatic synthesis of photoactive DNA. Evans, R.K., Johnson, J.D., and Haley, B.E. (1986). Proc. Natl. Acad. Sci. USA 83:5382-5386. (3461438)

⁶Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. Evans, R.K. and Haley, B.E. (1987) Biochemistry 26:269-276. (3548818)

⁷The cell-specific transcription factor PTF1 contains two different subunits that interact with the DNA. Roux, E., Strubin, M., Hagenbuchle, and Wellauer, P.K. (1989). Genes and Develop. 3:1613-1624. (2612907)

⁸Photoaffinity labeling of terminal deoxynucleotidyl transferase. 2. Identification of peptides in the nucleotide binding domain. Evans, R.K., Beach, C.M., and Coleman, M.S. (1989). Biochemistry 28:707-712. (2713339)

⁹Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA. Lee, D.K., Evans, R.K., Blanco, J., Gottesfeld, J., and Johnson, J.D. (1991). J. Biol. Chem. 266:16478-16484. (1885581)

¹⁰BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA. Blackwell, J.L. and Briton, M.A. (1995). J. Virol. 69:5650-5658. (7637011)

¹¹Identification of the Escherichia coli 30S ribosomal subunit protein neighboring mRNA during initiation of translation. Dontsova, O., Rosen, K.V., Bogdanova, S.L., , Skripkin, E.A., Kopylov, A.M. and Bodanov, A.A. (1992). Biochimie 74:363-371. (1379079)

¹²Characterization of a photoaffinity analog of UTP, 5-azido-UTP for analysis of the substrate binding site on E. coli RNA polymerase. Woody, A-Y.M., Evans, R.K., and Woody, R.W. (1988). Biochem. Biophys. Res. Comm. 150:917-924. (2449209)

¹³The L protein of vesicular stomatitis virus transcription complexes is specifically photolabelled by 5-azido-uridine 5'-triphosphate, an analogue of the RNA polymerase substrate uridine 5'-triphosphate. Hammond, D.C., Evans, R.K. and Lesnaw, J.A. (1992). J. Gen. Virol. 73:61-66. (1309862)

¹⁴Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain. Farrar, Y.J., Evans, R.K., Beach, C.M., and Coleman, M.S. (1991). Biochemistry  30:3075-3082. (2007141)

¹⁵ Photoaffinity labeling of the Klenow fragment with 8-azido-dATP. Rush, J. and Konigsberg, W.H. (1990). J. Biol. Chem. 265:4821-4827. (2180951)

¹⁶Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate. Woody, A-Y.M., Vader, C.R., Woody, R.W., and Haley, B.E. (1984). Biochemistry 23:2843-2848. (6380575)

¹⁷Photoaffinity labeling of rotavirus VP1 with 8-azido-ATP: identification of the viral RNA polymerase. Valenzuela, S., Pizarro, J., Sandino, A.M., Vasquez, M., Fernandez, J., Hernandez, O., Patton, J. and Spencer, E. (1991). J. Virol. 65:3964-3967. (1645806)

¹⁸Photoaffinity labeling of influenza virus RNA polymerase PB1 subunit with 8-azido GTP. Asano, Y., Mizumoto, K., Maruyama, T. and Ishihama, A. (1995). J. Biochem. 117:677-682. (7629040)

Technical Information or General Inquiries

Contact Dr. Anjan Bhattacharyya, Ph.D.
Radiochemicals Laboratory Director
E-mail: ajbhatta@Photoprobe.com