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 develop 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 fluorescence 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 can 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 modifications 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. ? ? ?
| Owczarzy, Richard; You, Yong; Groth, Christopher L et al. (2011) Stability and mismatch discrimination of locked nucleic acid-DNA duplexes. Biochemistry 50:9352-67 |
| You, Yong; Tataurov, Andrey V; Owczarzy, Richard (2011) Measuring thermodynamic details of DNA hybridization using fluorescence. Biopolymers 95:472-86 |
