Light controlled synthesis of nucleic acids

• Author(s): Pinheiro, Andre Alexandre Cotta Guerra Vidal
• Date: 2010
• Persistent ID: http://hdl.handle.net/10362/5760
• Origin: Repositório Institucional da UNL
• Document Type: Doctoral Thesis
• Language: English
• Advisor(s): Lima, Carlos; Baptista, Pedro

Description

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia The main objective of this thesis was the design and development of a system for the enzymatical synthesis of nucleic acids controlled by light. The overall concept is based on the functionalization of nucleotides with photoremovable protecting groups (or caged‐nucleotides), that cannot be recognized as substrates by the polymerases. Upon light absorption, the photo‐protecting group is cleaved and the nucleotide released, thus being incorporated in a growing nucleic acid chain. The specific release of the desired nucleotide, from a nucleotide mixture,is achieved functionalizing each type of nucleotide with a different caging group, presenting a distinct absorption spectrum. Through irradiation with monochromatic light, the specific nucleotide can be released unambiguously, leading to its incorporation. The irradiation sequence would, ultimately, define the sequence of the strand being formed. In order to overcome the template‐directed DNA (or RNA) synthesis, a template‐independent DNA polymerase was used – Terminal deoxynucleotidyl Transferase. Derivatives of 4‐methylcoumarin were chosen as photoremovable protecting groups and the successful synthesis of caged‐nucleotides was achieved. The photophysical and photochemical characterization of [7‐diethylaminocoumarin‐4‐yl]methyl phosphate (DEACM‐P) was performed. A dependence of the DEACM‐P photochemistry on pH was found, and a new model for 4‐methylcoumarin derivatives photochemistry was proposed. This model accounts for the hydroxyl concentration in the 4‐hydroxymethylcoumarin photoproduct formation. The photophysics and photochemistry characterization of P3‐[7‐diethylaminocoumarin‐4‐yl]methyl adenosine triphosphate (DEACM‐ATP), P3‐ [7‐diethylaminocoumarin‐4‐yl]methyl guanosine triphosphate (DEACM‐GTP), P3‐[7‐methoxycoumarin‐4‐yl]methyl adenosine triphosphate (MCM‐ATP) and P3‐[7‐methoxycoumarin‐4‐yl]methyl guanosine triphosphate (MCM‐ATP) was performed. The DEACM and MCM groups present absorption spectra in different regions (λmax = 390 nm and 325 nm, respectively), allowing for the desired selective irradiation and cleavage.Caged‐nucleotides were applied to in vitro transcription reactions. When the nucleotide was functionalized with a coumarin derivative, only residual RNA product formation could be detected. After irradiation, full size specific transcription product was obtained, showing that light can be used to activate the synthesis of nucleic acids. Both DEACM and MCM derivatives were used as caging groups, presenting similar behavior. Both ATP and GTP were successfully used as actuators for the lightcontrolled synthesis of RNA, although no transcription was attained when DEACM‐GTP was used. The light‐activated incorporation of nucleotides in a growing nucleic acid strand was successfully performed using the T7 RNA Polymerase and the Terminal deoxynucleotidyl Transferase. It was found that the 7‐diethyl 4‐hydroxymethylcoumarin (DEACM‐OH) photoproduct presented an inhibitory effect over the T7 RNA Polymerase, but that the inhibition could be partially suppressed through the addition of β‐cyclodextrin to the reaction.