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8-Substituted-2’-Deoxyguanosines as Internal Probes for DNA Folding and Energy Transfer


Dumas, A. 8-Substituted-2’-Deoxyguanosines as Internal Probes for DNA Folding and Energy Transfer. 2011, University of Zurich, Faculty of Science.

Abstract

Secondary structures adopted by nucleic acids might play important roles in diverse cellular processes. Developing efficient tools for their detection can provide means to elucidate their biological roles. This work presents our efforts to develop fluorescent nucleobase analogs, 8-substituted-2’-deoxyguanosines, that can serve as internal probes for the detection of G quadruplexes structures. This dissertation consists of 8 chapters, including 2 introductory chapters, 5 chapters presenting research results and one chapter of experimental procedures.

An overview of relevant aspects of DNA chemistry and conformations is presented in Chapter 1. The reader is introduced to some general characteristics of G-quadruplex structures and to strategies available for fluorescent labeling of nucleic acids.

In Chapter 2 the spectroscopic properties and potential applications of nearly all fluorescent purine analogs reported to date are reviewed.
Our strategy towards the development of new fluorescent purine analogs with improved characteristics focuses on the elaboration of 8-substituted-2’-deoxyguanosines.

In Chapter 3 the synthetic strategy developed to prepare these compounds is described. Further photophysical and conformational analysis of the resulting fluorescent nucleosides are presented in this chapter.

The structural impact and fluorescence properties of the 8-substituted guanosine derivative 8-(2-pyridyl)-2’-deoxyguanosine (2PyG) upon its incorporation into G quadruplex and duplex structures is presented in Chapter 4. A comparative study with the commonly used fluorescent nucleobase 2-aminopurine is presented. These results were published as part of: Dumas, A. and Luedtke, N. W. ChemBioChem 2011, in press, doi: 10.1002/cbic.201100214.

2PyG can act as an effective energy acceptor for unmodified guanine residues in oligonucleotides. In Chapter 5, the use of 2PyG to quantify this phenomenon in G rich sequences is presented. This property was exploited to detect DNA folding, and elucidate the direct role played by cations bound in the central cavity of G-quadruplex structures. Highly efficient energy transfer reactions are detected in G-quadruplex structures but not in duplex or single stranded DNA. Parts of these results were published in: Dumas, A. and Luedtke, N. W. J. Am. Chem. Soc., 2010, 132, 18004-18007.

In Chapter 6 two alternative fluorescent guanosine analogs, 8-(2-phenylethenyl)-2’-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2’-deoxyguanosine (4PVG) for the quantification of energy transfer and detection of G-quadruplex folding are introduced. These probes provide some potential advantages over 2PyG. Parts of these results were published in: Dumas, A. and Luedtke, N. W. Nucleic Acids Res., 2011, in press,
doi:10.1093/nar/gkr281.

2PyG is unique among all reported fluorescent purine analogs as it has the ability to selectively bind metals. Chapter 7 describes how this property can be exploited to site specifically localize metal ions at the N7 position of guanosine residues.

Detailed experimental procedures are described in Chapter 8. An extensive collection of circular dichroism and fluorescence spectra is provided in the Appendixes section.

Abstract

Secondary structures adopted by nucleic acids might play important roles in diverse cellular processes. Developing efficient tools for their detection can provide means to elucidate their biological roles. This work presents our efforts to develop fluorescent nucleobase analogs, 8-substituted-2’-deoxyguanosines, that can serve as internal probes for the detection of G quadruplexes structures. This dissertation consists of 8 chapters, including 2 introductory chapters, 5 chapters presenting research results and one chapter of experimental procedures.

An overview of relevant aspects of DNA chemistry and conformations is presented in Chapter 1. The reader is introduced to some general characteristics of G-quadruplex structures and to strategies available for fluorescent labeling of nucleic acids.

In Chapter 2 the spectroscopic properties and potential applications of nearly all fluorescent purine analogs reported to date are reviewed.
Our strategy towards the development of new fluorescent purine analogs with improved characteristics focuses on the elaboration of 8-substituted-2’-deoxyguanosines.

In Chapter 3 the synthetic strategy developed to prepare these compounds is described. Further photophysical and conformational analysis of the resulting fluorescent nucleosides are presented in this chapter.

The structural impact and fluorescence properties of the 8-substituted guanosine derivative 8-(2-pyridyl)-2’-deoxyguanosine (2PyG) upon its incorporation into G quadruplex and duplex structures is presented in Chapter 4. A comparative study with the commonly used fluorescent nucleobase 2-aminopurine is presented. These results were published as part of: Dumas, A. and Luedtke, N. W. ChemBioChem 2011, in press, doi: 10.1002/cbic.201100214.

2PyG can act as an effective energy acceptor for unmodified guanine residues in oligonucleotides. In Chapter 5, the use of 2PyG to quantify this phenomenon in G rich sequences is presented. This property was exploited to detect DNA folding, and elucidate the direct role played by cations bound in the central cavity of G-quadruplex structures. Highly efficient energy transfer reactions are detected in G-quadruplex structures but not in duplex or single stranded DNA. Parts of these results were published in: Dumas, A. and Luedtke, N. W. J. Am. Chem. Soc., 2010, 132, 18004-18007.

In Chapter 6 two alternative fluorescent guanosine analogs, 8-(2-phenylethenyl)-2’-deoxyguanosine (StG) and 8-[2-(pyrid-4-yl)-ethenyl]-2’-deoxyguanosine (4PVG) for the quantification of energy transfer and detection of G-quadruplex folding are introduced. These probes provide some potential advantages over 2PyG. Parts of these results were published in: Dumas, A. and Luedtke, N. W. Nucleic Acids Res., 2011, in press,
doi:10.1093/nar/gkr281.

2PyG is unique among all reported fluorescent purine analogs as it has the ability to selectively bind metals. Chapter 7 describes how this property can be exploited to site specifically localize metal ions at the N7 position of guanosine residues.

Detailed experimental procedures are described in Chapter 8. An extensive collection of circular dichroism and fluorescence spectra is provided in the Appendixes section.

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Additional indexing

Item Type:Dissertation
Referees:Luedtke N W, Siegel J S, Sigel R K O
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:3 October 2011
Deposited On:05 Mar 2012 13:41
Last Modified:07 Dec 2017 12:12
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&CON_LNG=GER&func=find-b&find_code=SYS&request=006847557

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