Journal of Photochemistry and Photobiology A: Chemistry 213 (2010) 147–151 Contents lists available at ScienceDirect Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem Use of cyclodextrins as scavengers of inhibitory photo-products in light controlled in vitro synthesis of RNA A. Vidal Pinheiro a,b , J. Conde b , A.J. Parola a , J.C. Lima a , P.V. Baptista b,∗ a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal b CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal article info Article history: Received 22 March 2010 Received in revised form 14 May 2010 Accepted 23 May 2010 Available online 31 May 2010 Keywords: Caged nucleotides Coumarin In vitro transcription Cyclodextrin RNA abstract 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 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 eval- uated the use of molecular scavengers to sequester DEACM-OH formed after irradiation. Determination of association constants of coumarin with -cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of -cyclodextrin in transcription reaction was also assessed. Results show that -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. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Controlled temporal and spatial release of biomolecules from photolabile precursors, commonly known as caged molecules, is of extreme relevance as a tool for bio-molecular studies [1–4]. The cage (chemical modification with a photolabile protecting group) renders the molecule of interest biologically inactive; upon irra- diation with light of a suitable wavelength, the biologically active molecule is released, generating a time-controlled burst in concen- tration with tight spatial control. For this purpose, the selected cage molecule ought to fulfill several critical conditions: (i) the efficiency of uncaging should be high in order to avoid long irradiation times and deleterious effects to the biological samples; (ii) the rate of uncaging must be faster than the process under study, i.e. the rate at which the biomolecule is released should not be the rate limit- ing step; (iii) easy to synthesize; and (iv) the resulting by-products should not hamper the desired reaction. We have previously described the use of ATP nucleotides caged with [7-(diethylamino)coumarin-4-yl]methyl (DEACM-ATP) for light-controlled in vitro transcription reactions, where the ∗ Corresponding author. Fax: +351 21 2948530. E-mail address: pmvb@fct.unl.pt (P.V. Baptista). quantity of RNA being polymerized could be controlled through DEACM-ATP irradiation [5]. Coumarin derivatives present high molar absorption coefficients, high photochemical quantum yields and absorptions in the visible region of the spectrum (<420 nm), with the advantage of fast photocleavage kinetics and wave- length tunability through changes in the position and/or nature of the chemical residues attached to the coumarin moiety [6–9]. In most aspects, coumarin cages, and in particular DEACM, follow the above mentioned selection requirements. However, following irradiation, high concentrations of photo-by-product 7- diethylamino-4-hydroxymethylcoumarin (DEACM-OH) inhibited the transcription reaction [5]. This effect was attributed to the poor coumarin solubility in water leading to the partitioning of the hydrophobic DEACM-OH molecule to the T7 RNA Polymerase, causing inhibition. To circumvent the solubility issue, hydroxyl or acetate groups can be added to the coumarin moiety that due to their anionic characteristics at physiological pH increases consid- erably the coumarin water solubility [10,11]. Although it might constitute a robust process of reducing the inhibition, it requires additional synthesis steps, on top of an already complex synthetic route. Here, we present an alternative supramolecular approach to decrease the inhibitory effect of water insoluble DEACM photo-by- products in enzymatic reactions – using -cyclodextrin molecules 1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotochem.2010.05.014