COBRE: UID: Pilot: Targeting Of Double Stranded DNA Using Locked Nucleic Acids (FY 2009)
Sequence-specific targeting of double stranded DNA (dsDNA) with exogenous nucleic acid probes remains an essentially unsolved challenge of biological chemistry. This is unfortunate since a general dsDNA targeting methodology potentially would yield highly rewarding outcomes including development of: powerful diagnostic probes for fluorescence in situ hybridization (FISH) studies, research tools for gene modulation, and potential drug candidates that may prevent formation of a disease-related protein at a very early stage (improved antigene strategy). The proposed project seeks to further refine and throughly characterize a recently discovered dsDNA targeting methodology, which is based on intercalator-modified nucleic acids. More specifically, DNA duplex probes with +1 interstrand arrangements of pyrene-functionalized 2'-amino-¿-L-LNA (Locked Nucleic Acid) monomers are very unstable (intercalator-induced duplex unwinding), while each of the strands exhibit an enormous affinity toward complementary DNA as a consequence of precise positioning of the intercalator in the duplex core. This thermal advantage is exploited as a driving force to facilitate fast sequence-specific recognition of dsDNA targets at physiologically relevant salt concentrations, which conveniently can be followed by a fluorescence assay. Herein, we propose to optimize this molecular recognition process by fine-tuning the chemical building blocks via attachment of more efficient intercalators (e.g., perylene, coronene or ethidium bromide analogs) and/or by synthesizing the more convenient O2'-alkylated RNA or N2'-functionalized 2'-amino DNA analogs of these building blocks. Further we propose to carry our an extensive series of biophysical characterization experiments (thermal denaturation, fluorescence, strand exchange, NMR, molecular modeling, etc ) to utilize this information for rational design of novel more efficient analogs, and to evaluate the full diagnostic/therapeutic potential of this methodology.