A simple, versatile and site-specific labeling technique permits the incorporation of fluorophores or other agents into nucleic acids. The backbone labeling procedures proposed in the previous application have been achieved during the last award period. In the present proposal, we will exploit these procedures in a number of areas. (i) The ability to easily tether a specific reporter group to a DNA sequence provides the foundation for the development of a series of DNA diagnostics. These materials will exploit complementary base pairing interactions for sequence recognition (and targeting) while delivering the diagnostic agent. In this proposal we will synthesize and study the properties of a nucleic acid hybridization probe. This conjugate will tether a minor groove binding fluorophore (Hoechst 33258). Upon location of the target DNA sequence, hybridization generates the minor groove structure in which the tethered fluorophore binds and the fluorescence signal is generated. (ii) Photoaffinity materials can be a powerful group of agents for understanding complex structures or processes. We will employ two types of backbone photoaffinity agents, the perfluoroazides and a simple benzophenone derivative, to study a protein-DNA complex (the trp repressor-operator) and to study a hammerhead ribozyme. (iii) Fluorescence resonance energy transfer (FRET) is an important biophysical technique that permits the measurement of distances from approximately 10 - 60 Angstroms. We will continue the FRET studies initiated during the previous application to study both the trp repressor- operator complex and the hammerhead ribozyme. Many of the DNA/RNA sequences used in (ii) and (iii) are similar but will vary in the agent (photoaffinity label or fluorophore) tethered to the sequence.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037065-09
Application #
2178672
Study Section
Medicinal Chemistry Study Section (MCHA)
Project Start
1986-07-01
Project End
1997-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
9
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Boston College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
045896339
City
Chestnut Hill
State
MA
Country
United States
Zip Code
02467
Gianolio, D A; McLaughlin, L W (2001) Tethered naphthalene diimide intercalators enhance DNA triplex stability. Bioorg Med Chem 9:2329-34
Wiederholt, K; Rajur, S B; McLaughlin, L W (1997) Oligonucleotides tethering Hoechst 33258 derivatives: effect of the conjugation site on duplex stabilization and fluorescence properties. Bioconjug Chem 8:119-26
O'Donnell, M J; Rajur, S B; McLaughlin, L W (1995) Synthesis and properties of a Hoechst-like minor-groove binding agent tethered to an oligodeoxynucleotide. Bioorg Med Chem 3:743-50
Fidanza, J A; Ozaki, H; McLaughlin, L W (1994) Functionalization of oligonucleotides by the incorporation of thio-specific reporter groups. Methods Mol Biol 26:121-43
Ozaki, H; McLaughlin, L W (1992) Fluorescence resonance energy transfer between specific-labeled sites on DNA. Nucleic Acids Symp Ser :67-8
Hall, K B; McLaughlin, L W (1992) Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex. Nucleic Acids Res 20:1883-9
Ozaki, H; McLaughlin, L W (1992) The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer. Nucleic Acids Res 20:5205-14
Conway, N E; McLaughlin, L W (1991) The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA. Bioconjug Chem 2:452-7
Loontiens, F G; McLaughlin, L W; Diekmann, S et al. (1991) Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents. Biochemistry 30:182-9
Hall, K B; McLaughlin, L W (1991) Properties of a U1/mRNA 5' splice site duplex containing pseudouridine as measured by thermodynamic and NMR methods. Biochemistry 30:1795-801

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