TY - JOUR
T1 - Use of cyclodextrins as scavengers of inhibitory photo-products in light controlled in vitro synthesis of RNA
AU - Vidal Pinheiro, André
AU - Conde, João
AU - Parola, António Jorge
AU - Lima, João Carlos
AU - Baptista, Pedro Viana
N1 - 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.
PY - 2010/6/25
Y1 - 2010/6/25
N2 - 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 μM [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 β-cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of β-cyclodextrin in transcription reaction was also assessed. Results show that β-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.
AB - 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 μM [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 β-cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of β-cyclodextrin in transcription reaction was also assessed. Results show that β-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.
KW - Caged nucleotides
KW - Coumarin
KW - Cyclodextrin
KW - In vitro transcription
KW - RNA
UR - http://www.scopus.com/inward/record.url?scp=77955713074&partnerID=8YFLogxK
U2 - 10.1016/j.jphotochem.2010.05.014
DO - 10.1016/j.jphotochem.2010.05.014
M3 - Article
AN - SCOPUS:77955713074
SN - 1010-6030
VL - 213
SP - 147
EP - 151
JO - Journal of Photochemistry and Photobiology A: Chemistry
JF - Journal of Photochemistry and Photobiology A: Chemistry
IS - 2-3
ER -