Is it possible to study the kinetic parameters of interaction between PNA and parallel and antiparallel DNA by stopped-flow fluorescence?
Peptide nucleic acids (PNAs) are among the most interesting and versatile artificial structural mimics of nucleic acids and exhibit peculiar and important properties (i.e. high chemical stability, and a high resistance to cellular enzymes and nucleases). Despite their unnatural structure, they are able to recognize and bind DNA and RNA in a very high, specific and selective manner. One of the most popular, easy and reliable method to measure the stability of PNA-DNA hybrid systems is the melting temperature but the thermodynamic data are obtained using a big quantity of materials failing to provide information on the kinetics of the interaction. In the present work, the PNA decamer 6, with the TCACTAGATG sequence of nucleobases, and the corresponding fluorescent PNA-FITU (fluorescein isothiourea) decamer 8 were synthesized with standard manual Boc-based chemistry. The interaction of the PNA-FITU with parallel and antiparallel DNA has been studied by stopped-flow fluorescence, which is proposed as an alternative technique to obtain the kinetic parameters of the binding. The great advantage of using the stopped-flow technique is the possibility of studying the kinetics of the PNA-DNA duplex formation in a physiological environment. In particular, fluorescence stopped-flow technique has been exploited to compare the affinity of two PNA-DNA duplexes since it can discriminate between parallel and antiparallel DNA binding.
Department of Chemistry and NIS Interdepartmental Centre, University of Torino, Via P. Giuria 7, I-10125 Torino, Italy. Electronic address: nadia.barbero@unito.it.
N
N
Barbero
Department of Chemistry, University of Milano, Via C. Golgi 19, I-20133 Milano, Italy. Electronic address: silvia.cauteruccio@unimi.it.
S
S
Cauteruccio
Department of Chemistry, University of Milano, Via C. Golgi 19, I-20133 Milano, Italy.
P
P
Thakare
Department of Chemistry, University of Milano, Via C. Golgi 19, I-20133 Milano, Italy.
E
E
Licandro
Department of Chemistry and NIS Interdepartmental Centre, University of Torino, Via P. Giuria 7, I-10125 Torino, Italy.
G
G
Viscardi
Molecular Biotechnology and Health Sciences Department, University of Torino, Via Quarello 15, 10135 Torino, Italy.
S
S
Visentin
D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D020135:Peptide Nucleic Acids; D004247:DNA
Affinity; Binding; DNA; Kinetics; PNA; Stopped-flow fluorescence