Abstract

A nucleoside analogue which could base pair equally well with all of the natural bases in a nucleic acid duplex would find extensive application as a tool for molecular biology. As a primer component for the polymerase chain reaction (PCR), a universal (or wildcard) nucleoside could be used in nucleic acid amplification techniques including the generation of randomized DNA pools as a method for protein engineering. 1. The design, synthesis, and physicochemical properties of nucleoside analogues derived from five-membered ring heterocycles will be studied. From molecular modeling studies, target molecules were selected which would pair with natural nucleic acids in duplex or triplex structures with minimal distortion of the helices. Most of the analogues contain five-membered ring heterocycles which have substituents capable of hydrogen bonding in different configurations to each of the nucleic acid bases. The primary goal of this research is to find nucleoside analogues that may function as wild-cards in commonly used molecular techniques involving DNA amplification, replication, and sequencing. 2. Oligonucleotides will be constructed in which the modified nucleosides are incorporated at contiguous and non-contiguous multiple sites. The effect of the modifications on duplex structure will be investigated by Tm measurements and NMR spectroscopy. 3. Modified nucleoside phosphoramidites and triphosphates, and CPG linked modified nucleosides will be prepared and provided to collaborators to test their utility in a variety of systems. 4. Synthetic oligonucleotides containing five member heterocyclic purine/pyrimidine mimics will be analyzed as primers for PCR reactions. Each of these heterocycles have been designed to adopt potential conformations that allow for hydrogen bond recognition of all four naturally occurring purine and pyrimidine bases. 5. Nucleotide triphosphates that mimic the recognition of both purine and pyrimidine bases will be used as substrates for a variety of DNA polymerases.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM053155-03
Application #
2685067
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1996-04-01
Project End
1999-06-30
Budget Start
1998-04-01
Budget End
1999-06-30
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
Purdue University
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907

  Publications

Knudsen, Giselle M; Davis, Brandon M; Deb, Shirshendu K et al. (2008) Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman. Bioconjug Chem 19:2212-20
Deb, Shirshendu K; Davis, Brandon; Knudsen, Giselle M et al. (2008) Detection and relative quantification of proteins by surface enhanced Raman using isotopic labels. J Am Chem Soc 130:9624-5
Deb, Shirshendu K; Davis, Brandon; Ben-Amotz, Dor et al. (2008) Accurate concentration measurements using surface-enhanced Raman and deuterium exchanged dye pairs. Appl Spectrosc 62:1001-7
Fang, Shiyue; Guan, Yousheng; Blatchley 3rd, Ernest R et al. (2008) Conjugation of (E)-5-[2-(Methoxycarbonyl)ethenyl]cytidine to hydrophilic microspheres: development of a mobile microscale UV light actinometer. Bioconjug Chem 19:592-7
Adelfinskaya, Olga; Davisson, V J; Bergstrom, Donald E (2005) Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs. Nucleosides Nucleotides Nucleic Acids 24:1919-45
Adelfinskaya, Olga; Nashine, Vishal C; Bergstrom, Donald E et al. (2005) Efficient primer strand extension beyond oxadiazole carboxamide nucleobases. J Am Chem Soc 127:16000-1
Paul, Natasha; Nashine, Vishal C; Hoops, Geoffrey et al. (2003) DNA polymerase template interactions probed by degenerate isosteric nucleobase analogs. Chem Biol 10:815-25
Fang, Shiyue; Bergstrom, Donald E (2003) Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides. Nucleic Acids Res 31:708-15
Wu, Weidong; Bergstrom, Donald E; Davisson, V Jo (2003) A combination chemical and enzymatic approach for the preparation of azole carboxamide nucleoside triphosphate. J Org Chem 68:3860-5
Fang, Shiyue; Guan, Yousheng; Blatchley 3rd, Ernest R et al. (2003) Development of a nucleoside analog UV light sensor. Nucleosides Nucleotides Nucleic Acids 22:703-5

Showing the most recent 10 out of 14 publications