Lincoln Scott - 8-Azaguanine reporter of purine ionization states in structured RNAs.

Version 1

      Publication Details (including relevant citation   information):

      J Am Chem   Soc. 2007 Mar 21;129(11):3426-32. Epub 2007 Feb 28.

      Abstract:

      The fluorescent nucleotide analogue   8-azaguanosine-5'-triphosphate (8azaGTP) is prepared easily by in   vitro enzymatic synthesis methods. 8azaGTP is an efficient   substrate for T7 RNA polymerase and is incorporated specifically   opposite cytosine in the transcription template, as expected for   a nucleobase analogue with the same Watson-Crick hydrogen bonding   face as guanine. 8-Azaguanine (8azaG) in oligonucleotides also is   recognized as guanine during ribonuclease T1 digestion. Moreover,   replacement of guanine by 8azaG does not alter the melting   temperature of base-paired RNAs significantly, evidence that   8azaG does not disrupt stacking and hydrogen bonding   interactions. 8azaGTP displays a high fluorescent quantum yield   when the N1 position is deprotonated at high pH, but fluorescence   intensity decreases significantly when N1 is protonated at   neutral pH. Fluorescence is quenched 10-fold to 100-fold when   8azaG is incorporated into base-paired RNA and remains   pH-dependent, although apparent pKa values determined from the pH   dependence of fluorescence intensity shift in the basic   direction. Thus, 8azaG is a guanine analogue that does not   perturb RNA structure and displays pH-dependent fluorescence that   can be used to probe the ionization states of nucleobases in   structured RNAs. A key application will be in determining the   ionization state of active site nucleobases that have been   implicated in the catalytic mechanisms of RNA enzymes.

      Address (URL): http://www.ncbi.nlm.nih.gov/pubmed/17326637