We recently reported on the use of caged nucleotides to attain full control of enzymatic polymerization of RNA solely by light. In the absence of light no RNA formation was possible due to the efficient caging by the coumarin moiety: after irradiation, caged ATP was released with quantitative precision and RNA polymerization was resumed. As photolabile protecting group [7-(diethylamino)coumarin-4-yl]methyl] (DEACM) was used due to its high absorbance in the visible region of the spectrum, fast deprotection kinetics and absence of radical intermediates. However, the 7-diethylamino-4-hydroxymethylcoumarin photo-product (DEACM-OH) was shown to inhibit the transcription reaction for concentrations higher than 30 mu M [5]. This inhibition has been associated with poor water solubility, which is commonly dealt with via cumbersome chemical modifications of the protecting moiety. To overcome inhibition, we evaluated the use of molecular scavengers to sequester DEACM-OH formed after irradiation. Determination of association constants of coumarin with beta-cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of beta-cyclodextrin in transcription reaction was also assessed. Results show that beta-cyclodextrin can be successfully used as scavenger as it increases the DEACM-OH threshold concentration for inhibition, amplifying the efficiency of light controlled in vitro transcription. (C) 2010 Elsevier B.V. All rights reserved.
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Cited Reference Count: 22
Pinheiro, A. Vidal Conde, J. Parola, A. J. Lima, J. C. Baptista, P. V.
Conde, Joao/F-2231-2011; Parola, A. Jorge/F-4048-2010; Caparica, cqfb_staff/H-2611-2013; Baptista, Pedro/A-1237-2009; REQUIMTE, AL/H-9106-2013; Chaves, Pedro/K-1288-2013; Lima, Joao/F-3658-2010
Conde, Joao/0000-0001-8422-6792; Parola, A. Jorge/0000-0002-1333-9076; Baptista, Pedro/0000-0001-5255-7095; Lima, Joao/0000-0003-0528-1967
Fundacao para a Ciencia e Tecnologia [SFRH/BD/24276/2005, SFRH/BD/62957/2009]; REQUIMTE; CIGMH [PTDC/SAU-BEB/66511/2006]
The authors would like to acknowledge Dr. Cesar Laia for fruitful suggestions. This work was supported by Fundacao para a Ciencia e Tecnologia - A.V.P. (SFRH/BD/24276/2005) and J.C. (SFRH/BD/62957/2009), REQUIMTE, CIGMH, PTDC/SAU-BEB/66511/2006.
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