The goal of the proposed research is to scale up the production and manufacture of high-value ribonucleoside triphosphates containing stable isotope labels. The use for these products is in biomolecular studies of RNA structure using Nuclear Magnetic Resonance (NMR) spectroscopy. The market for these products is structural biology groups in academic and industrial laboratories engaged in structure determination or NMR screening of RNA. Stable isotope labeling with 13C, 15N, and 2H is a requirement for application of multidimensional NMR studies for structural biology of macromolecules. Existing methods for labeling of RNA and DNA suffer from three main drawbacks. First, the production of isotopically labeled nucleotides is complex and time consuming and difficult to carry out in individual laboratories. Second, it is extremely expensive, since the materials for a single sample can cost many thousands of dollars. Third, the existing methods only permit uniform isotope labeling, while there is considerable advantage to selective isotope labeling. Recently, we have developed technology that will ameliorate or eliminate these three drawbacks. The technology involves the efficient single-pot synthesis of labeled ribonucleotides from labeled precursors using a series of enzymatic transformations. In order to make these reagents widely available to the structural biology community, we propose to optimize and scale up production of these reagents for commercial distribution.
| Schultheisz, Heather L; Szymczyna, Blair R; Scott, Lincoln G et al. (2008) Pathway engineered enzymatic de novo purine nucleotide synthesis. ACS Chem Biol 3:499-511 |
| Da Costa, Carla P; Fedor, Martha J; Scott, Lincoln G (2007) 8-Azaguanine reporter of purine ionization states in structured RNAs. J Am Chem Soc 129:3426-32 |
