The Martinez group uses biochemistry to dissect the RNAi mechanism in human cells. Besides classical fractionation techniques, the group has already developed a procedure to affinity-purify RISC, the RNA-Induced Silencing Complex, and is continuously elaborating specific tools for the identification of enzymatic and non-enzymatic factors along the RNAi pathway (Martinez, J. et al., 2002, Cell 110: 563). The Martinez group is currently identifying miRNA-binding proteins in order to reveal the miRNA's mechanism of action. Moreover, in close collaboration with the Dickson laboratory, both groups have established a robust protocol to follow the expression of miRNAs by in situ hybridization using LNA (locked nucleic acids). This technique will become a method of choice to evaluate disease models linked to aberrant miRNA expression. The Martinez group is also undertaking a classical biochemical approach to identify other enzymatic activities in the RNAi pathway. It has been shown that only upon phosphorylation of 5'- hydroxyl termini, siRNA duplexes become competent for RNAi. siRNAs lacking the 5' phosphate become rapidly phosphorylated in HeLa cytoplasmic extracts by a yet unidentified kinase that may also be required to maintain the phospho-modification intracellularly. Currently the kinase activity has been partially purified over several chromatographic steps, and its biochemical properties and activity characterized in great detail.
