Over the past two decades, I have been interested in developing RNA targeted drug therapy. I have led the overall experimental approach focused on developing antisense technology at Ionis. I have contributed to the evolution of Ago2 based RNA reduction, led significant research that resulted in the formation of Regulus to pursue antimir therapeutics. Additionally, I have been involved in creating mass spectrophotometric methods to evaluate small molecule RNA interactions.
My current research interests focus on understanding the molecular mechanisms by which antisense drugs work. In the last several years, we have identified the major intracellular proteins with which ASOs interact. We have shown that 52 proteins appear to interact with ASOs and determine the intracellular fate and activity of ASOs. We are developing a detailed understanding of the structure activity relationships that influence ASO protein binding and the structural domains in proteins that interact with ASOs. We have developed new approaches that support the rapid characterization of ASO protein interactions.
We continue to be interested in human RNase H1 having cloned and characterized both human RNase H1 and H2 some years ago. We have recently identified the functions of RNase H1 in somatic cells and shown that RNase H1 and topoisomerase 1 are often redundant and inversely regulated. We have created two viable mouse liver RNase H1 knockout strains and are using those systems to better understand the rolls RNase H1 plays in ASO activity, the toxicity of ultra high affinity ASOs and liver homeostasis.
Having characterized RNase H1 and the proteins that interact with it, we have recently identified the nuclear and cytoplasmic pathways that degrade the RNase H1 induced RNA fragments. As a result, we are now studying the enzymological properties of XRN1 and XRN2 as well as 3
Additionally, we have studied Ago2 mediated target RNA reduction. We have reported a number of studies showing the interaction of various oligonucleotides with Ago2 and studied a number of mechanisms of off target effects. We have shown that some dsRNAs can work by altering poly A site utilization. Finally, we have pioneered the use of ss modified RNA like ASOs to activate Ago2.
Several years ago we developed a unique system that supports the seamless evaluation of ASO interactions and effects on a structured
We are now beginning to look in depth at intracellular transport processes and pathways that affect ASOs. We are also exploring how membrane composition and lipid transport pathways alter ASO affects.
Finally, we are keenly interested in expanding the number of mechanisms by which ASOs can be used to increase protein production. We have shown that by altering upstream open reading frames and translation inhibitory elements and other mechanisms, we can specifically enhance production of many individual proteins.
Molecular Mechanisms of ASO Publications
RNase H1
Vickers, T.A. and Crooke, S.T. The rates of the major steps in the molecular mechanism of RNase H1-dependent antisense oligonucleotide induced degradation of RNA. Nucleic Acids Res 43:8955-8963, 2015.
Vickers, T. and Crooke, S.T. Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms. PLos One 9:e108625, 2014.
Vickers, T.A., Freier, S.M., Bui, H.H., Watt, A. and Crooke, S.T. Targeting of repeated sequences unique to a gene results in significant increases antisense oligonucleotide potency. PLos One 9:e110615, 2014.
Wu, H., Sun, H., Liang, X., Lima, W.F. and Crooke, S.T. Human RNase H1 is associated with Protein P32 and is involved in mitrochondrial pre-rRNA processing. PLos One 8:e71006, 2013.
Liang, X., Vickers., T.A., Guo, S. and Crooke, S.T. Efficient and specific knockdown of small non-coding RNAs in mammalian cells and in mice. Nucleic Acids Research 39:1-17, 2011.
Lima, W.F., Wu, H. and Crooke, S.T. The RNase H Mechanism. In Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition, Crooke, ST. (ed) Baco Raton, FL, Chapter 2, 47-74, 2008.
Lima, W.F. et al. The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis. Mol Pharmacol 71:73-82, 2007.
Lima, W.F. et al. Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate. Mol Pharmacol 71:83-91, 2007.
Lima, W.F. et al. Human RNase H1 uses a tryptophan and two lysines to position the enzyme at the 3’-DNA/5’-RNA terminus of the heteroduplex substrate. J Biol Chem 278:49860-49867, 2004.
Wu, H. et al. Determination of the Role of the Human RNase H1 in the Pharmacology of DNA-like Antisense Drugs. J Biol Chem 279:17181-9, 2004.
Lima, W.F. et al. The structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis. J Biol Chem 279:36317-36326, 2004.
Vickers, T.A. et al. Efficient Reduction of Target RNAs by siRNA and RNase H-Dependent Antisense Agents: A Comparative Analysis. J Biol Chem 278:7108-7118, 2003.
Lima, W.F. et al. Human RNase H1 activity is regulated by a unique redox switch formed between adjacent cysteines. J Biol Chem 278:14906-14912, 2003.
Wu, H., Lima, W.F. and Crooke, S.T. Investigating the structure of human RNase H1 by site-directed mutagenesis. J Biol Chem 276:23547-23553, 2001.
Lima, W.F. and Crooke, S.T. Human RNase H. Methods Enzymol 341:430-440, 2001.
Wu, H., Lima, W.F. and Crooke, S.T. Properties of cloned and expressed Human RNase H. J Biol Chem 274:28270-28278, 1999.
Wu, H., Lima, W.F. and Crooke, S.T. Molecular cloning and expression of cDNA for human RNase H. Antisense and Nuc Acid Drug Dev 8:53-61, 1998.
Lima, W.F. and Crooke, S.T. Binding affinity and specificity of Escherichia coli RNase H1: Impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids. Biochemistry 36:390-398, 1997.
Lima, W.F., Mohan, V. and Crooke, S.T. The influence of antisense oligonucleotide-induced RNA structure on E. coli RNase H1 activity. J Biol Chem 272:18191-18199, 1997.
Lima, W.F. and Crooke, S.T. Cleavage of single-strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1. J Biol Chem 272:27513-27516, 1997.
Crooke, S.T. et al. Kinetic characteristics of E. coli RNase H1: Cleavage of various antisense oligonucleotides-RNA duplexes. Biochem J 312:599-608, 1995.
AGO2
T.P. et al. Identification of Metabolically Stable 5’-Phosphate Analogs That Support Single Stranded siRNA Activity. Nucleic Acid Res 43:2993-3001, 2015.
Liang, X., Hart, C.H. and Crooke, S.T. Transfection of siRNAs can alter miRNA levels and trigger non-specific protein degradation in mammalian cells. Biochim Biophys Acta 1829:455-468, 2013.
Prakash, T. et al. Lipid-Like Delivery Molecules Improve Activity of Single-Stranded siRNA and Gapmer Antisense Oligonucleotides in Animals. ACS Chem Biol 8:1402-6, 2013.
Liang, X. and Crooke, S.T. RNA helicase A is not required for RISC activity. BBA – Gene Regulatory Mechanisms 1829:1092–1101, 2013.
Lima, W. et al. Single-stranded siRNAs Activate RNAi in Animals. Cell 150:883-894, 2012.
Corey, D. et al. Single-Stranded RNAs Act Through RNAi to Potently and Allele-Selectively Inhibit Huntingtin Expression. Cell 150:5, 895-908, 2012.
Lima, W.F et al. Human Dicer binds short single-strand and double-strand RNA with high affinity and interacts with different regions of the nucleic acids. J Biol Chem 284:2535-2548, 2009.
Lima, W.F., Wu, H., Sun, H., Murray, H.M. and Crooke, S.T. Binding and Cleavage Specificities of Human Argonaute2. J Biol Chem 284:25653-25663, 2009.
Vickers, T.A. et al. Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1. Nucleic Acids Research 37:6927-6941, 2009.
Vickers, T., Lima, W.F., Nichols, J.G. and Crooke, S.T. Reduced levels of Ago2 expression result in increased siRNA competition in mammalian cells. Nucleic Acids Res 35:6598-610, 2007.
Vickers, T.A. et al. Efficient Reduction of Target RNAs by siRNA and RNase H-Dependent Antisense Agents: A Comparative Analysis. J Biol Chem 278:7108-7118, 2003.
Wu, H., Xu, H., Miraglia, L.J. and Crooke, S.T. Human RNase III is a 160-kDa protein involved in preribosomal RNA processing. J Biol Chem 275:36957-36965, 2000.
Wu, H., MacLeod, R., Lima, W.F., and Crooke, S.T. Identification and partial purification of human double-strand RNase activity: a novel terminating mechanism for oligoribonucleotide antisense drugs. J Biol Chem 273:2532-2542, 1998.
SHIFTING POLY-A SITES OR ALTERING RNA SPLICING
Vickers, T. and Crooke, S.T. Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms. PLos One 9:e108625, 2014.
Vickers, TA, Sabripour, M and Crooke, ST. U1 Adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP. Nucleic Acids Research 39:1-12, 2011.
Vickers, T. and Crooke, S.T. siRNAs targeted to certain polyadenylation signals promote specific, RISC-independent degradation of messenger RNAs. Nucleic Acid Res 40:1-12, 2011.
Hodges, D. and Crooke, S.T. Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides. Mol Pharmacol 48:905-918, 1995.
OTHER NUCLEASES
Lima, W.F. and Crooke, S.T. Preparation and use of ZFY-6 Zinc Finger Ribonuclease. Methods Enzymol 341:490-500, 2001.
Lima, W.F. and Crooke, S.T. Highly efficient endonucleolytic cleavage of RNA by a Cys2His2Zinc finger peptide. Proc Natl Acad Sci 96:10010-10015, 1999.
SUBCELLULAR DISTRIBUTION
Liang, X., Sun, H., Shen, W. and Crooke, S.T. Identification and characterization of intracellular proteins that bind phosphorothioate-containing oligonucleotides. Nucleic Acid Res 43:2927-2945, 2015.
Shen, W., Liang, X.H., Sun, H. and Crooke, S.T. 2′-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF. Nucleic Acid Res, ePub April 2015.
Shen, W., Liang, X. and Crooke, S.T. Phosphorothioate oligonucleotides can displace NEAT1 RNA and form nuclear paraspeckle-like structure. Nucleic Acid Res 14:8648-8662, 2014.
Liang, X., Shen, W., Sun, H., Prakash, T.P. and Crooke, S.T. TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic Acid Res 42:7819-7832, 2014.
GENERAL PRINCIPLES
Liang, X., Sun, H., Shen, W. and Crooke, S.T. Identification and characterization of intracellular proteins that bind phosphorothioate-containing oligonucleotides. Nucleic Acid Res 43:2927-2945, 2015.
Lima, W., Vickers, T., Nichols, J., Li, C. and Crooke, S.T. Defining the factors that contribute to on-target specificity for antisense oligonucleotides. PLos One 9:e101752, 2014.
Vickers, T.A., Freier S.M., Bui H.H., Watt, A. and Crooke S.T. Targeting of repeated sequences unique to a gene results in significant increases antisense oligonucleotide potency. PLos One 9:e110615, 2014.
Crooke, S.T., Vickers, T., Lima, W.F. and Wu, H. Mechanisms of Antisense Drug Action, an Introduction. In Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition, Crooke, ST. (ed), Baco Raton, FL, CRC Press, Chapter 1, 3-46, 2008.
Miraglia, L., Watt, A.T., Graham, M.J. and Crooke, S.T. Variations in mRNA content have no effect on the potency of antisense oligonucleotides. Antisense and Nuc Acid Drug Dev 10:453-461, 2000.
Crooke, S.T. Molecular Mechanisms of Antisense Drugs: Human RNase H’s. Antisense and Nuc Acid Drug Dev 9:377-379, 1999.
Crooke, S.T. Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489:30-42, 1999.
Crooke, S.T. Molecular mechanisms of antisense drugs: RNase H. Antisense and Nuc Acid Drug Dev 8:133-134, 1998.
Crooke, S.T. Basic Principles of Antisense Therapeutics. In Antisense Research and Application. Handbook of Experimental Pharmacology, Stanley T. Crooke (ed), Springer-Verlag, Berlin Heidelberg, 131, 1-50, 1998.