Making Better siRNA Tools
Dharmacon (Boulder, CO) provides tools for RNAi practitioners as well as strategies for minimizing false positive and false negative results. Acquired by Fisher Scientific in 2004, Dharmacon was originally founded to commercialize new technologies for synthesizing RNA oligonucleotides developed at the University of Colorado at Boulder.
Anastasia Khvorova, Ph.D., Dharmacon's vp of research, talks about the major challenges facing siRNA users and how Dharmacon's technology has addressed them.
Dr. Khvorova says that most of their customers use siRNA for target validation, knocking out one gene at a time to study pathways and determine the effect on phenotype. Alternatively, she says, many large pharmaceutical clients with greater resources look at broad collections of genes such as groups of kinases, GPCRs, or the current collection of "druggable genes".
The challenge in performing large screens of this sort is to minimize the number of false negatives that occur when siRNAs fail to downregulate the expression of the intended target gene.
"In principle," she says, "It's quite possible to predict how functional an siRNA will be based on its nucleotide sequence. siRNAs function through the RNA Interference Silencing Complex, and some siRNAs are better substrates for this complex than others."
"While there are exceptions to the rules (some genes are easier to silence than others), certain parameters such as thermodynamic stability profiles are shared amongst all functional siRNA."
Dharmacon's approach to the problem of false negatives was to develop a highly predictive algorithm that screens target sequences for traits that are common amongst functional siRNA.
"We use proprietary algorithms to identify the best siRNA target sites within any given gene sequence." The four top-scoring siRNAs are then provided as a pool (as well as individ ual duplexes) to each customer.
According to Dr. Khvorova, the second major challenge in using siRNAs is minimizing false positives. She says that that the "highly specific" nature of siRNAs is something of a myth. Partial sequence homology between an siRNA and unintended targets can lead to the down-regulation of dozens of genes, and expression changes of these "off-targets" can result in false positives during phenotypic screens.
The problem of off-targeting can be partially solved by mimicking nature. Inside the cell, long double-stranded RNA is processed by Dicer into pools. While the total concentration of siRNA directed against the primary target is high (and thus produces strong silencing), the concentration of any individual duplex within the pool is low, thus minimizing the potential for off-targeting.
Dharmacon has adopted the pooling strategy in their attempts to eliminate off-targeting, and further addressed the problem by identifying a group of chemical modifications that can be applied to any siRNA to increase specificity.
Developed in collaboration with Merck and Rosetta and reported at the recent "Keystone Symposium on RNAi," Dharmacon believes it has created a "new generation of siRNA reagents".
Their recently developed Reverse Transfection (RTF) technology will allow rapid, accurate, and cost-effective high throughput knockdown of every gene in the human genome, according to the company.
Dharmacon says that its RNAi product innovations should enable researchers in academic and small biotechnology laboratories to join the ranks of the big pharma labs in being able to cost-effectively perform genome-wide phenotypic screens with minimal or no false positives.