Many developments related to DNA diagnostics or therapeutics will require the detection of specific DNA or RNA sequences with extremely high sensitivity. The development of reporter groups and the ability to place them in multiple or specific positions in a nucleic acid sequence will not only facilitate detection by procedures which are rapid and inexpensive but will also allow the study of nucleic acids or nucleic acid protein complexes which are responsible for the nucleic acid related diseases or for their cure. This proposal will concentrate in two areas: (1) The incorporation of multiple fluorescent labels into nucleic acid sequences containing phosphorothioate diesters in a procedure we designate """"""""post-assay"""""""" labeling, that is, the label is only attached after DNA sequencing or hybridization procedures. """"""""Post-assay"""""""" labeling by the procedures described allows the detection of nucleic acid sequences in the low femtomolar (10 15 mole) range without the use of sophisticated electronic detection. With this level of sensitivity, fluorescent labeling offers an alternative to the use of radioisotopes, thereby eliminating the associated health and environmental hazards. (2) Using phosphorothioate diesters, reporter groups can be placed at specific locations for the study of the structure and dynamics of unusual nucleic acids as well as protein - nucleic acid complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037065-07
Application #
3292018
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1986-07-01
Project End
1993-07-31
Budget Start
1992-07-01
Budget End
1993-07-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Boston College
Department
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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