Exploring the small RNA transcriptome, a key challenge

Small RNA are involved in numerous biological process and linked to a wide range of conditions. For example miRNA can be used for detection of cancers from blood samples and are used for cancer classification. They are involved in the regulation of the messenger RNA translation.

The small RNA transcriptome is a vast world rich in information. There are not only miRNA but other small RNA that can also be used for diagnostics. For example, Repetto et al in 2015 (doi: 10.1186/s12916-015-0489-y) discovered novel small RNA in blood, s-RNY1-5p as a diagnostic biomarker for atherosclerosis-related diseases. The authors used small-RNA sequencing with the innovative CleanTag technology.

Capping is a required protection in 5′ of the mRNA that is necessary for biological activity and stability into the cells. There are already several methods of capping using mCap, ARCA or enzymatic methods. Unfortunately, they are limited in efficiency (such as mCap and ARCA), and can be costly (such as the enzymatic methods). Trilink Biotechnologies, the modified nucleic acid experts, have developped a new, far more efficient technology called CleanCap available in Europe via tebu-bio.

Decidedly, producing and purifying a protein is a form of art.

Indeed, each recombinant protein (rec. protein) to be produced in vitro has specific characteristics and singularities that make its production and purification most definitely challenging. In this post, I’d like to share and review a few tips and basics for optimal design of experiment when producing functional recombinant proteins and working on protein purification.

Sequence engineering and chemical modification of CAS9 mRNA

Extract from poster “Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”.

For transgenesis and therapeutic perspectives, vector-free delivery of the CAS9 for gene editing is highly preferable. It avoids the risk of hitting the genome due to the vector integration. Directly using CAS9 mRNA is a top option.

Furthermore, as a new promising class of therapeutic biologics, messenger RNA are the subject of numerous studies.

This year, Matthew Porteus’s and Trilink’s teams revealed key and practical results at the ASGC with a poster you can download below.

Notably, there is a large screen of the mRNA CAS9 modifications based on the indel formation. This interesting comparison includes also CAS9 RNP and reveals the high performance of the CleanCap U-depleted 5-moU CAS9 mRNA. We can also note that for the CleanCap CAS9 mRNA HPLC purification is not required.

“Maximizing Translation of Cas9 mRNA Therapeutics by Sequence Engineering and Chemical Modification”
Download your copy of the poster here.