Case Number 115104 - Codon optimized uridine-specific ribonuclease MC1 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 ribonuclease MC1 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. Among other potential issues the current invention solves the following problems:

• Lack of nucleoside-specific ribonucleases for RNA structural analysis: This invention’s construct generates a uridine-specific RNase that complements the commercially available guanosine-specific RNase T1 and cytidine/uridine selective RNase A. In particular, RNase MC1 should have a greater utility than RNase A, which cleaves RNA at a greater frequency due to its lack of specificity.

• RNase MC1 should be useful for structural analysis of GC-rich RNAs: GC-rich RNAs, which tend to be more stable than AT-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. GC-rich RNAs have, by definition, a lower frequency of adenosine and uridine residues. Thus, the uridine-specific MC1 RNase should yield larger oligomers from GC-rich RNAs, which provide a greater amount of structural information within a given experiment.

• Ambiguous isotope effects in mass spectrometric analysis: Improved mass spectrometry analysis for RNA modification mapping: uridine differs from cytidine by 1 Da (O 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 MC1 will yield oligomers containing a single uridine in the sequence at the 5’-terminus of each digestion product. Moreover, the number of cytidines should also be more easily determined based on accurate mass measurements and prior sequence reconstruction challenges will be eliminated.

The technology of this invention is superior to the current state of the art because it 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.

The MC1 nuclease can also have further hi-tech and low-tech applications open to the labs that are interested in comparing the profiles of uridine modifications in the transcriptome (but do not necessarily employ mass spec based analysis). Here, the comparison of MC1 digest of in vitro transcript with that of the native RNA (can be tRNA, rRNA, etc.) would allow straightforward mapping of [m5U] and [s4U] and potentially other bulky modifications on a transcriptome wide scale by RNA-seq approaches or simple PAGE depending on the complexity of sample.

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

A provisional patent application (62/151,640) has been filed on this technology.