The fundamental building blocks of the genetic code (DNA, RNA and proteins) are all subject to chemical modifications following their synthesis. In DNA and proteins, such modifications have been extensively studied for decades, and are known to play critical roles in cell physiology and pathology; As such, they also serve as critical achilles heels for therapeutic intervention. In recent years it has been discovered that RNA, too, is subjected to a key, potent post-transcriptional modification, in the form of N6-Methyladenosine (m6A). Recent studies have demonstrated that this ‘epitranscriptomic’ mark plays fundamental roles in regulation of metabolism, stem cell self-renewal, and metastasis, and consistently its dysregulation is implicated in a variety of human cancers including leukemia, glioblastoma and hepatocellular carcinoma. The potential of m6A both as a novel cancer diagnostic and as a drug target have spurred immense interest in the biotech and pharma sectors, with the goals of developing m6A-based diagnostic and therapeutic approaches.
At the basic science level, an ‘epitranscriptome’ community has evolved, encompassing hundreds of labs addressing the roles, functions and mechanisms of action of m6A in diverse systems and model organisms. Key goals in this community are to unravel the diverse roles and mechanisms of action of m6A. However, a crucial limitation in the field has been the lack of quantitative methodologies for monitoring m6A levels. Current tools can only provide a qualitative sense of m6A being present at a specific site. This makes research progress slow, inaccurate, and expensive, because m6A is not an ‘all or none’ modification, but instead can occur at one site at only a small fraction of transcripts (‘low level’), whereas at a different site it might occur at nearly all transcripts (‘high level’). Moreover, these levels can change as part of a cellular response or in the context of diseases. For understanding m6A function, for the development of proper diagnostic tools and for prioritizing diseases and disease states most likely to benefit from therapeutic targeting of the methylation complex, it is thus of crucial importance to be able to systematically measure the levels at which m6A is present, and to quantitatively monitor changes in these levels across disease progression. Therefore, a quantitative tool for assessing m6A levels is vital.
In our prior work, we developed MAZTER-seq, a first quantitative readout of m6A levels, relying on a methylation-sensitive ribonuclease (RNase). Specifically, our approach relies on the ability of a bacterially derived RNA cleaving enzyme to cleave only at sites that are not methylated. If a site is methylated, the enzyme can no longer cleave at this site. The relative frequency of cleavage at each site - which we can measure on the basis of a genomic, sequencing-based protocol - thus provides an inverse measurement of the methylation levels. This metric allows to accurately quantify modification levels, and represents the first systematic approach for quantitatively interrogating of m6A levels. The major benefits are in the comprehensive, accurate and swift quantitative readings, performed with low starting material. Thus, it can be directly applied for boosting the development of biomarkers and therapeutics targeting the methylation machinery.
In the context of this proof-of-concept grant, we set to further develop this protocol in a manner both enhancing its scale and its accuracy, and to simultaneously explore the possibility of commercializing this approach, which is of broad utility in the scientific, pharma and biomedical communities. We have been able to make progress on both ends, yet still have not been able to fully overcome a technical barrier, that at the moments limits both the utility and the patentability of this protocol. In ongoing work we hope to overcome these barriers.
