Predicting stability of nucleic acid duplexes containing chemical modifications
DESCRIPTION (provided by applicant): Synthetic DNA and RNA oligomers are important tools for biological and medical assays including polymerase chain reactions, sequencing, DNA microarrays, antisense technology and RNA interference. Oligomers are generally
used in experiments where they anneal to complementary sequences. Accurate predictions of annealing processes and duplex stability are therefore critical for design of such experiments. Nearest-neighbor model and thermodynamic parameters have been highly
successful in predicting stability of native DNA and RNA duplexes. Dozens of useful chemical modifications of nucleic acids that give new properties to synthetic oligomers have been introduced (e.g., phosphorothioates, 2-O'-methyl RNA, locked nucleic acids
(LNA), biotin, 5-nitroindole). However, these modifications are not widely used because their thermodynamic parameters are often unknown and effects of modifications on duplex stability cannot be accurately predicted. Our long-term goal, then, is to devel
op new, faster methods to measure thermodynamic parameters and to use these methods to determine parameters for various chemical modifications of nucleic acids, so that design of oligomers containing modifications is improved. We propose a novel fluorescen
ce melting method that could provide thermodynamic parameters at least 10 times faster than traditional ultraviolet spectroscopic and calorimetric methods. The approach takes advantage of high-throughput real-time PCR systems (e.g. Bio-Rad iCycler) that ca
n be modified to obtain equilibrium melting profiles. To demonstrate feasibility of the novel method in Phase I, we will determine unknown thermodynamic parameters for locked nucleic acids. We are requesting funding for the specific aims of: i) development
of high-throughput fluorescence method for measurements of thermodynamic parameters, ii) application of the fluorescence method to determine unknown thermodynamic parameters for locked nucleic acid modifications. Synthetic DNA and RNA oligomers containing
chemical modifications (e.g., locked nucleic acids, phosphorothioate DNAs, 2'-O-methyl RNAs) are used for both diagnosis and treatment of disease. We propose to develop a fluorescence melting method that could promptly determine effects of these modificat
ions on DNA and RNA duplex stability. Such improved understanding of duplex stability for chemically modified nucleic acids will help design more accurate and efficient diagnostic tests and medical experiments.
Small Business Information at Submission:
INTEGRATED DNA TECHNOLOGIES, INC.
INTEGRATED DNA TECHNOLOGIES 1710 COMMERICAL PARK CORVALVILLE, IA 52241
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