|Specificity of highly potent miRNA inhibitors|
Barbara Robertson, Andrew Dalby, Yuriy Fedorov, Jon Karpilow, Anastasia Khvorova1, Devin Leake, Annaleen Vermeulen
miRNA inhibitors are invaluable tools for elucidating the roles of miRNAs. However, potent inhibitors may also affect other miRNAs. To understand the potential cross-reactivity of miRNA inhibitors, various miRNA inhibitor designs were systematically tested. We demonstrate that mismatches both within and outside the seed region of the miRNA interfere with inhibition. Our findings indicate that features important for natural miRNA target recognition are also important for inhibitor specificity.
|Alternative miRNA design for therapeutic RNAi applications|
Anja van Brabant Smith, Barb Robertson, Annaleen Vermeulen, Christina Yamada, Angela Reynolds, Anastasia Khvorova, Devin Leake
For in vivo applications, the design of miRNA inhibitors and miRNA mimics must be optimized for stability and potency. However, stabilized miRNA mimic molecules can lose functionality compared to standard miRNA mimic molecules due, in part, to the activity of the stabilized passenger strand acting as a miRNA inhibitor. We discuss how mismatches affect the activity of the stabilized miRNA mimics, perhaps by generating a passenger strand that is less functional as an inhibitor molecule.
|Cas9 driven by an optimal promoter improves gene editing in eukaryotic cell lines when paired with synthetic crRNA and tracrRNA|
Amanda Haupt, Emily Anderson, Žaklina Strezoska, Hidevaldo Machado, Shawn McClelland, Maren Mayer, Adam Rocker, Annaleen Vermeulen, Amanda Birmingham, Melissa Kelley, Anja Smith
Presented here are results on the efficiency of using synthetic crRNA and tracrRNA to introduce gene editing events when co-transfected with a plasmid expressing Cas9. We explored the use of antibiotic and fluorescence activated cell sorting (FACS) methods for enrichment of cells that have undergone gene editing, and the use of multiple promoters to increase efficiency of gene editing with Cas9 and synthetic tracrRNA and crRNAs.
|Specificity and functionality of microRNA inhibitors|
Barbara Robertson, Andrew Dalby, Jon Karpilow, Anastasia Khvorova, Devin Leake and Annaleen Vermeulen
Our findings indicate that features important for natural miRNA target recognition also appear to be important for inhibitor specificity. Understanding the specificity of inhibitors allows for better interpretation of inhibitor activity in endogenous systems.
|Identification of microRNA targets using microRNA modulation techniques and gene expression arrays|
Emily M. Anderson, Maren Mayer, Kevin Sullivan, Barbara Robertson, Žaklina Strezoska, Annaleen Vermeulen, and Devin Leake
By examining the overlap of messages down-regulated by miRNA mimics and up-regulated by miRNA inhibitors, we robustly identify miRNA-regulated messages, many of which have canonical seed matches and some which are not identied by standard target prediction programs.
|siRNA Screening: Development of Hit Stratification Strategies|
Žaklina Strezoska, Annaleen Vermeulen, Emily M. Anderson, Anja Smith, Devin Leake
This poster compares different approaches to hit stratification and validation after an initial screen. Standard siRNA reagents deconvoluted from a pooled set of four were compared to a pooled set of four specificity enhanced reagents. High confidence hits were similar. To explore the validity of low confidence hits, a chimeric approach was used whereby a gene-specific seed sequence was introduced into a non-targeting siRNA scaffold. This work resulted in new hit stratification strategies.
|The Chemical Synthesis of Long and Highly Modified RNA using 2'-ACE Chemistry|
Xiaoqin Cheng, Kristina Larson, Letitia Kwok, David Mierzejewski, Shawn Begay, Randy Rauen, Kelly Grimsley, Kaizhang He, Michael Delaney, David Kitchen, Amanda Haas, Melissa Kelley, Anja van Brabant Smith
Dharmacon has previously developed a novel RNA synthesis chemistry making RNA synthesis as reliable, accessible and of comparable quality as routinely observed in DNA synthesis.
|Droplet-on-Demand Platform for Biochemical Screening & Drug Discovery|
L.D. van Vliet1*, F. Gielen1, A. Sinha2, B.T. Koprowski3, J.B. Edel4, X.Niu5, A.J. deMello3, F. Hollfelder1, & J. Motschman2
To demonstrate droplet on demand applications towards study of biological entities encapsulated in nanoliter droplets. Interfacing a droplet on demand platform with microfluidic chips allows for merging and dilution of droplets. This feature is applied to encapsulate yeast cells (S. cerevisiae) and multicellular organisms (C. elegans).
|Direct Targets Identification of a Bioactive Compound|
Sylvain Blanc, Paul Bradley, Marie-Edith Gourdel, Michael Cholay, Gisèle Guimèse, Mike Mckenzie, George Nasi, Jean-Christophe and Barbara Ruggiero
Identifying protein partners of a small bioactive molecule is of great
interest in many aspects of life sciences and specifically in the drug
discovery and development process cycle. It is a support to (i) decipher
the mechanism of action after for example a “High Content” screening,
(ii) study “off-target” effects, (iii) adjust therapeutic indications and
clinical regimens of a drug and (iv) consider drug repositioning.