PROJECT SUMMARY
Protein synthesis in mammalian cells depends not only on the transcription of RNA from the DNA
genome, but also on the stability of that RNA and its rate of translation. A class of ~21 nucleotide RNAs
known as ‘microRNAs’ play critical roles in regulating protein synthesis by recognizing complementary
sequences in protein-coding RNA molecules and causing either RNA cleavage or inhibition of translation.
MicroRNA regulation plays key roles in nearly every studied human physiological system, and mis-
regulation of individual microRNAs or global microRNA processing has been linked to cancer, cardiac and
kidney disease, viral infection response, and many other diseases. Identifying the targets of individual
microRNAs provides an essential insight into the functional biological role of each microRNA. Furthermore,
as functional regulatory molecules microRNAs are now being highly pursued for their use as therapeutic
agents, where the ability to directly map microRNA targets is required to assay both proper on-target
binding as well as low off-target interactions. However, current methods are lacking, as they have either
high false-positive rates, lack the ability to assign targets to individual microRNAs, or do not scale to
profiling all (particularly low-abundance) microRNAs with quantitative accuracy.
Recently we developed the enhanced CLIP-seq (eCLIP) methodology, with 1000-fold improved
efficiency of generating high-throughput sequencing libraries from RBP profiling experiments, enabling
highly robust and reproducible RBP target profiling through the incorporation of paired size-matched inputs.
Eclipse has successfully developed eCLIP as a highly profitable contract service product, and now has an
eCLIP kit in beta testing. Here we will develop a specialized variant of the eCLIP method for unambiguous
mapping of microRNA targets transcriptome-wide in the following three aims:
1. Validate unambiguous direct profiling of miRNA targets with chimeric eCLIP (chim-eCLIP).
2. Validate simplified chim-eCLIP method for commercialization.
3. Conversion of chimeric eCLIP into a structured and well-documented kit format.
Eclipse Bio is an ideal candidate to perform the aims described above due to our expertise in genomics and
computational biology, particularly in RNA processing and profiling RNA binding protein targets. The three
aims above will enable microRNA target mapping to be performed in a standard method by all biomedical
researchers in academia and industry, and create a rigorous standard for validating specificity of therapeutic
microRNAs. The ability to properly assess therapeutic miRNA-like molecules will provide significant benefits
to researchers studying microRNA regulation in various biological contexts and drug companies developing
RNA therapies in the clinic. Additionally, in San Diego we are close to many research institutes and biotechs
doing RNA research that can provide scientific and commercialization expertise and assistance.