Case Number 115107 - Codon optimized cytidine-specific ribonuclease Cuvastin gene

Contact: Doug Nienaber
Phone: 513-558-3098

Description:  Drs. Patrick Limbach and Balasubrahmanyam Addepalli have discovered a recombinant plasmid encoding a codon-optimized synthetic Cusativin gene that holds promise to increase research productivity in the area of RNA structural analysis which has been inhibited by the lack of nucleoside-specific enzymes (RNases).

To improve RNA structural analysis, additional RNases with nucleoside specificity that complements what is available from RNase T1, RNase A and RNase U2 are desirable. Advantages of a new nucleoside-specific RNase include, but are not limited to, the following:

• Lack of nucleoside-specific ribonucleases for RNA structural analysis: This construct generates a cytidine-specific RNase that complements the commercially available guanosine-specific RNase T1 and cytidine/uridine selective RNase A. In particular, Cusativin should have a greater utility than RNase A, which cleaves RNA at a greater frequency due to its lack of specificity.

• Cusativin should be useful for the structural analysis of G-rich RNAs: G-rich RNAs are difficult to characterize with RNase T1, as the higher frequency of guanosine residues yields smaller oligomers that do not always yield useful scientific data on the parent RNA structure. The cytidine-specific Cusativin should yield larger oligomers from G-rich RNAs, which will provide a greater amount of structural information within a given experiment.

• Cusativin yields information about C-rich regions of RNA structure: Cusativin at defined amounts cleave the phosphodiester bond at 3’-end of cytidine efficiently when it is followed by Adenosine or Guanosine or Uridine (C-A or C-G or C-U). However, the phosphodiester bond between two cytidines (C-C) is not cleaved efficiently, thereby, releasing the cytidine-rich region (for example, CCCCU) as oligonuclecotide (CCCC and U) instead of (just C and U). This enables the structure/sequence determination even in case of C-rich regions of RNA.

• Improved mass spectrometry analysis for RNA modification mapping: Uridine differs from cytidine by 1Da (0 versus NH) and the presence of C-13 isotopes, which are readily detected by mass spectrometry, can easily result in challenges in differentiating the number and sequence location of these pyrimidines. This challenge is particularly noteworthy for larger digestion products wherein the "all light" (C-12) isotope peak is no longer the most abundant. Digestion with Cusativin should yield oligomers containing a single cytidine in the sequence at the 3'-terminus of each digestion product. Moreover, the number of uridines should also be more easily determined based on accurate mass measurements and prior sequence reconstruction challenges will be eliminated.

This technology solves the above stated problems by overexpressing the protein in bacteria where bacterial cells could serve as molecular factories for mass production of target proteins. Employment of the recombinant nucleobase-specific enzyme could ensure higher predictability and reproducibility of the data besides simplifying the experimental procedures and analysis. It also provides solutions to various problems/issues listed above.

The target market for the invention is the RNA analysis/Transcriptomics market. RNA analysis is the study of the complete set of RNAs (transcriptome) encoded by the genome of a specific cell or organism at a specific time or under a specific set of conditions. RNA analysis has significant applications in the field of pharmaceuticals and biotechnology and is used in the drug discovery, life science research and clinical diagnostic applications. The global RNA analysis/Transcriptomics market was valued at $1.7 billion in 2013 and it is expected to reach $3.8 billion by 2019. The market is driven by technological advancements and the increase in government and private funding for research coupled with research and development investments by the biotechnology companies. Similarly, the application of RNA analysis in the field of biomarker discovery opens new growth opportunities with the rising preference of personalized medicine across the globe

A provisional patent application (62/151,546) has been filed on this technology.