RNA interference (RNAi)

RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing (PTGS) mechanism triggered in somatic mammalian cells by RNA duplexes of 21 – 23 nucleotides (nts) called small interfering RNAs or siRNAs. siRNA can be introduced into mammalian cells in a number of forms including duplexes formed from chemically synthesized RNA oligonucleotides and from single stranded RNA transcripts that form a hairpin stem loop structure, where the stem region corresponds to the siRNA sequence. These latter structures are known as short hairpin RNAs (shRNAs) that can also be expressed intracellularly from pol II or pol III promoters and can thus be cloned into plasmid and viral vectors. RNAi has been rapidly developed and adopted as a functional genomics tool in a wide range of species. RNAi based technologies have been adapted to allow for transient or stable knockdown of gene expression generation in cell lines and animals and have been developed for high throughput analysis of gene function in Caenorhabditis elegans and Drosophila. Several programs are in progress to generate siRNA sequences (both as synthetic siRNAs and as shRNAs) corresponding to most of the identified transcripts within the human genome. In addition, RNAi mediated by exogenously generated siRNAs is very closely related to the mechanism used by naturally occurring small non-coding RNAs termed miRNA to control the expression of endogenous gene expression. Recently, there is increasing evidence that aberrant miRNA expression can contribute to altered gene expression in cancer cells.

Synthetic siRNAs

Under a research collaboration agreement (RCA #2-18668-05) with Qiagen Inc. the Gene Silencing Section (GSS) is accessing synthetic siRNAs targeted towards genes associated with cancer and related process. Using these resources GSS is developing independent and collaborative studies in delineating the role of key pathways involved in cancer and related processes and for the identification and validation of new anti-cancer targets. As part of this work, GSS has developed an infrastructure for the characterization of the RNAi mediated by these siRNAs on a relatively high throughput basis at an RNA level (see below). Further, in collaboration with the Antibody Production and Purification Unit, CCR and Dr John Weinstein (Laboratory of Molecular Pharmacology, CCR) the GSS is developing highly sensitive and quantitative protein based assays to assess the overall consequence of RNAi at the RNA, protein and functional level. Optimized protocols for the transfer of synthetic siRNAs into a wide range of cancer relevant cell lines under development, including their utilization in 3D-cell culture models. In addition, the GSS has initiated a number of studies investigating the interaction of RNAi against specific genes and cellular drug responses as a means for understanding the mechanism of action of anticancer drugs and the characterization of their molecular target(s).

Access to RNAi characterization data (CCR Investigators only):

If you are a CCR Investigator, please follow this link to learn about access to RNAi characterization data.

Short hairpin RNAs

Short hairpin RNAs are single stranded RNA transcripts that form a hairpin stem loop structure where the stem region corresponds to the siRNA sequence. GSS has recently initiated a research program utilizing shRNA screens to study specific functional endpoints. Whereas transfections with siRNAs are typically transient, shRNAs can have the advantage of longer effects as cassettes can be expressed from integrating viral constructs. CCR investigators are encouraged to contact Drs. Natasha Caplen (ncaplen@mail.nih.gov) and Konrad Huppi (huppi@helix.nih.gov) to discuss specific collaborative projects and our current efforts to access large scale shRNA resources.

Micro RNAs

Micro RNAS (miRNAs) are small non-coding RNAs that control endogenous gene expression through interaction with a target transcript to either trigger transcript cleavage as seen for siRNAs or to block protein translation. GSS is developing a number of collaborative projects investigating the role of miRNAs in cancer. In collaboration with Dr. Robert Stephens, Advanced Biomedical Computer Center (NCI-Frederick, SAIC-Frederick Inc.) we are studying the bioinformatic analysis of miRNA target interactions and we are developing several collaborations with CCR investigators analyzing miRNA expression profiles.