Guanine-rich DNA and RNA sequences can fold into structures consisting of stacked guanine tetrads. These quadruplex structures are believed to play important roles in several biological contexts, including repressing transcription of the c-MYC oncogene, controlling alternative splicing of the pre-mRNA for hTERT, the reverse transcriptase component of telomerase and regulating the activity of telomerase. Molecules that bind to these quadruplex structures can serve as probes of biological function and potential therapeutics. ? ? High affinity PNA probes will be synthesized and targeted to DNA and RNA G-quadruplex structures. What distinguishes this proposal from standard antigene and antisense approaches to regulating gene expression is the fact that the PNAs will have homologous, rather than complementary, sequences to their targets. This strategy is feasible because G-rich PNAs are known to form hybrid quadruplexes with homologous DNA and RNA sequences. Hybridization thermodynamics and kinetics will be measured for PNA-DNA and PNA-RNA quadruplex formation using a combination of optical spectroscopy and surface plasmon resonance experiments. An important component of this project will be synthesizing various PNA designs will to optimize affinity and selectivity. Once this is achieved, PNAs will be synthesized and used to test binding to three G quadruplexes (2 DNA and 1 RNA) modeled on biologically relevant targets. Finally, PNAs will be introduced into mammalian cell culture to study inhibition of c-MYC transcription, alteration of hTERT pre-mRNA splicing and inhibition of telomerase activity. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM058547-06A1
Application #
7142752
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Lewis, Catherine D
Project Start
2000-04-01
Project End
2010-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
6
Fiscal Year
2006
Total Cost
$209,686
Indirect Cost
Name
Carnegie-Mellon University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Gupta, Anisha; Lee, Ling-Ling; Roy, Subhadeep et al. (2013) Strand invasion of DNA quadruplexes by PNA: comparison of homologous and complementary hybridization. Chembiochem 14:1476-84
Roy, Subhadeep; Zanotti, Kimberly J; Murphy, Connor T et al. (2011) Kinetic discrimination in recognition of DNA quadruplex targets by guanine-rich heteroquadruplex-forming PNA probes. Chem Commun (Camb) 47:8524-6
Lusvarghi, Sabrina; Murphy, Connor T; Roy, Subhadeep et al. (2009) Loop and backbone modifications of peptide nucleic acid improve g-quadruplex binding selectivity. J Am Chem Soc 131:18415-24
Roy, Subhadeep; Tanious, Farial A; Wilson, W David et al. (2007) High-affinity homologous peptide nucleic acid probes for targeting a quadruplex-forming sequence from a MYC promoter element. Biochemistry 46:10433-43
Marin, Violeta L; Armitage, Bruce A (2006) Hybridization of complementary and homologous peptide nucleic acid oligomers to a guanine quadruplex-forming RNA. Biochemistry 45:1745-54
Datta, Bhaskar; Bier, Mark E; Roy, Subhadeep et al. (2005) Quadruplex formation by a guanine-rich PNA oligomer. J Am Chem Soc 127:4199-207
Marin, Violeta L; Armitage, Bruce A (2005) RNA guanine quadruplex invasion by complementary and homologous PNA probes. J Am Chem Soc 127:8032-3
Marin, V; Hansen, H F; Koch, T et al. (2004) Effect of LNA modifications on small molecule binding to nucleic acids. J Biomol Struct Dyn 21:841-50
Datta, Bhaskar; Schmitt, Christoph; Armitage, Bruce A (2003) Formation of a PNA2-DNA2 hybrid quadruplex. J Am Chem Soc 125:4111-8
Kushon, S A; Jordan, J P; Seifert, J L et al. (2001) Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins. J Am Chem Soc 123:10805-13

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