They take the advantages of both aptamers and DNAzymes, which can recognize specific targets with high selectivity and catalyze multiple-turnover reactions for signal amplification, respectively, and have shown their great promise in many analytical applications.
AAG shows faster multiple-turnover reactions on single-stranded than on duplex DNA when the salt concentration is low [100 mM NaCl].
The time course data were fitted to the single exponential eq 4. The multiple-turnover reactions contained 60 μL of 200 μM 4-HB-CoA mixed with 60 μL of 20, 32, and 40 μM YdiI.
In contrast, for most of the white spots, no significant increase in spot intensity was observed when the enzyme concentration was increased 3-fold, indicating that those sequences are either extremely poor substrates or not substrates at all in terms of multiple-turnover reactions.
Next, we followed the two-step formation of Ile-tRNAIle under multiple-turnover reaction conditions by using P-labeled tRNA.
The 17% [O2]-benzoic acid observed for the single-turnover reaction is contrasted with the 4% [O2]-benzoic acid observed for the multiple-turnover reaction of benzoyl-CoA (750 μM) catalyzed by wild-type YdiI (45 μM), for which 6% is the predicted maximum value.
For the multiple-turnover reaction of YdiI-catalyzed hydrolysis of [C]-benzoyl-CoA, the final enzyme and substrate concentrations used were 10 and 50 μM, respectively, whereas for the single-turnover reaction, respective concentrations of 50 and 5 μM were employed.
Selections for enzymes can be performed indirectly (for binding of a transition-state analogue or mechanism-based inhibitor), and directly using either intramolecular single-turnover selections (e.g. with SELEX) or the normal (intermolecular, multiple turnover) mode of enzymatic reactions.
We performed time course experiments to monitor the progression of intermolecular strand-joining reactions under multiple turnover conditions using a 5'-FAM-labeled 31-mer RNA and a 17-mer 5'-adenylated DNA adapter.
