Isolation and purification of mRNA; identification of drug targets.
Isolation mRNA is the first step to sequencing expression sequence tags, which are the foundation of any genome sequencing project. Current methods of mRNA isolation depend on the presence of a 3' poly(A) tail. Therefore mRNAs that lack this 3' poly(A) tail or that have short poly(A) tails are not included in human genome microarrays. MRNAs with high degrees of structure also prevent poly(A) dependent methods for isolating mRNA. As a result, there are likely to be other biologically critical mRNAs that are unknown entities in the human genome and genome of pathogenic organisms.
An alternative approach to isolating eukaryotic mRNA is to bind a conserved chemical structural motif (e.g., 5' m7Gppp cap) that is present at the 5'-end of mRNA. However, the major limitation of this technique has been the low affinity of the protein used to bind the 5' m7Gppp cap. The current invention provides a more efficient method for isolating mRNA using mutant mRNA cap binding protein (4E), which has high affinity for the 5' m7Gppp cap.
Generally speaking, oligo(dT) purifications work very well for mRNAs, but work poorly when the 3' poly-A ends are short. In a recent PNAS paper describing this technology, 187 out of a total of 222 mRNAs were isolated in greater quantity using eIF4E than oligo(dT) columns, usually by a factor of at least two.