Generating short RNAs Long hairpin RNA yields population of several sequence specificities. Processed by Dicer. Tandem promoters expressing sense and antisense strands of siRNA in trans . Short hairpin RNA (shRNA), sense and antisense associating in cis . Processed by Dicer. Dykxhoorn D, Novina CD, Sharp PA (2003) Nature Reviews . 4: 457-467. In vitro generation, chemically synthesized DNA-vector mediated in vivo generation with RNA Polymerase family Imperfect duplex hairpin based on pre-miRNA structure. Processed by Dicer.
RNase III family characteristic cleavage
2n.t. 3’ overhang
Recessed 5’ phosphate
www.nature.com/focus/animations/animation/animation.htm Blaszczyk J, Tropea JE, Bubunenko M, Routzahn KM, Waugh DS, Court DL, Ji X (2001) Structure . 9: 1225-1236
RNA-Induced Silencing Complex
siRNA must be 5’- phosphorylated
Double-stranded siRNA is unwound; antisense strand complexes with RISC proteins.
Engineered Human Tumor Cells hTERT (protein component of telomerase) RAS V12 SV40 Large T oncoprotein SV40 Small T oncoprotein In collaboration with William Hahn and Stephen Lessnick, Dana-Farber Cancer Institute primary cell tumor cell
Sources of Small Molecules 1. Combinatorial Library - 20,000 compounds 2. NCI diversity set - 1,990 compounds 3. Annotated Compound Library - 2,036 compounds 24,026 compounds Test for > 50% inhibition in tumor cells 380 compounds > 4-fold tumor cell selectivity vs normal cells 9 compounds IC 50 primary IC 50 tumor
An Automated Process for Cell-Based Screening Chemical Libraries Plate Barcoding Plate Replication Sterile Cell Dispensing Assay Execution Data Analysis Root DE, Kelley BP Stockwell BR (2003) Detection of Systematic Errors in Spatial Arrays. J Biomolecular Screening ,8 (4) 393-398
Identification of Erastin-Binding Proteins B1 A6 B1 = immobilized inactive analog A6 = immobilized active analog erastin-specific binding protein In collaboration with Prolexys
Voltage-Dependent Anion Channel
Effect of Putative Target mRNA Knockdown on Erastin Sensitivity in Human Tumor Cells
BJeLR (engineered human tumor line) cells seeded at 200,000 cells/well in 384-well plates.
Anti-VDAC2 siRNA transfected via liposome complex with Oligofectamine.
Total RNA isolation.
Viability Assay (Alamar Blue)
Erastin-induced Cell Death in Tumor Cells Erastin
Chemical Genetic Screen
Stage 1 : Identification of small molecules that induce
Acknowledgements Collaborators William Hahn & Steve Lessnick Prolexys Pharmaceuticals Funding Burroughs Wellcome Fund National Cancer Institute Erastin Project Brent Stockwell Sonam Dolma Steve Flaherty Allison Martino