Synthetic agents that recognize specific sequences within chromosomal DNA are a promising strategy for controlling gene expression. During the previous funding period we described duplex RNAs, peptide nucleic acids (PNAs), PNA-peptides, and locked nucleic acids (LNAs) that target gene promoters and modulate expression. LNA bases contain a modified ribose with a methylene linkage between the 2'-oxygen and 4'-carbon and increase melting temperature values (Tm's) by as much as 100C per substitution. We focus this proposal on LNAs because LNAs have been the simplest and most effective tools for direct recognition of chromosomal DNA. Objectives. Our objective for the next funding period is to understand the potential of chromosome- targeted LNAs as research tools and leads for clinical development. Research Design. We will investigate the mechanism, optimization and applications of agLNAs.
In Aim 1 we will investigate the mechanism of agLNA-mediated recognition of nucleic acid targets and subsequent inhibition of gene expression inside cells. The environment surrounding gene promoters is a complex mix of RNA transcripts, proteins, and chromosomal DNA. Understanding the mechanism of recognition will help guide use of agLNAs for new applications and provide important basic insights into how synthetic oligomers affect cellular processes at gene promoters.
In Aim 2, we will characterize cellular uptake, improve delivery methods, and test chemically modified LNAs. The goal for this Aim is to identify the most straightforward and efficient combination of delivery protocol and chemical structure for achieving optimal inhibition with antigene oligonucleotides. This information will assist researchers hoping to use agLNAs and help guide the design of agLNAs for therapeutic applications.
In Aim 3 we propose novel strategies to use agLNAs to control cellular processes. Applications include use of agLNAs to probe accessibility of sequences within chromosomal DNA, targeting agLNAs to genes with bidirectional promoters (10 % of human genes), using agLNAs to manipulate expression of genes that are important biomedical targets, and synthesis and testing of agLNA-peptide conjugates designed to recruit transcription factors to gene promoters and activate gene expression. These experiments were chosen for their potential to expand the range of applications for agLNAs. Biomedical Relevance. There are currently few options for sequence-specific recognition of chromosomal DNA. agLNAs unlock access to chromosomal DNA and provide an alternative for addressing the difficult problem of achieving adequate potency and specificity in vivo. agLNAs will also provide useful tools for probing chromosome accessibility at specific sequences, information that may lead to a better understanding of replication, DNA repair, and gene expression.

Public Health Relevance

Chromosomal DNA encodes the information necessary to express proteins. Agents that target DNA have the potential to affect production of proteins involved in disease and may provide a new class of drugs. The goals of this proposal are to learn how recognition of DNA can be achieved and to use this knowledge to develop strategies for controlling the function of cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073042-08
Application #
8213759
Study Section
Special Emphasis Panel (ZRG1-GGG-J (10))
Program Officer
Fabian, Miles
Project Start
2005-02-01
Project End
2013-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
8
Fiscal Year
2012
Total Cost
$295,442
Indirect Cost
$107,263
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Gagnon, Keith T; Li, Liande; Janowski, Bethany A et al. (2014) Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading. Nat Protoc 9:2045-60
Gagnon, Keith T; Li, Liande; Chu, Yongjun et al. (2014) RNAi factors are present and active in human cell nuclei. Cell Rep 6:211-21
Aiba, Yuichiro; Hu, Jiaxin; Liu, Jing et al. (2013) Allele-selective inhibition of expression of huntingtin and ataxin-3 by RNA duplexes containing unlocked nucleic acid substitutions. Biochemistry 52:9329-38
Liu, Jing; Yu, Dongbo; Aiba, Yuichiro et al. (2013) ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy. Nucleic Acids Res 41:9570-83
Matsui, Masayuki; Chu, Yongjun; Zhang, Huiying et al. (2013) Promoter RNA links transcriptional regulation of inflammatory pathway genes. Nucleic Acids Res 41:10086-109
Liu, Jing; Pendergraff, Hannah; Narayanannair, K Jayaprakash et al. (2013) RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression. Nucleic Acids Res 41:8788-801
Gagnon, Keith T; Corey, David R (2012) Argonaute and the nuclear RNAs: new pathways for RNA-mediated control of gene expression. Nucleic Acid Ther 22:3-16
Hu, Jiaxin; Gagnon, Keith T; Liu, Jing et al. (2011) Allele-selective inhibition of ataxin-3 (ATX3) expression by antisense oligomers and duplex RNAs. Biol Chem 392:315-25
Gagnon, Keith T; Watts, Jonathan K; Pendergraff, Hannah M et al. (2011) Antisense and antigene inhibition of gene expression by cell-permeable oligonucleotide-oligospermine conjugates. J Am Chem Soc 133:8404-7
Hu, Jiaxin; Liu, Jing; Corey, David R (2010) Allele-selective inhibition of huntingtin expression by switching to an miRNA-like RNAi mechanism. Chem Biol 17:1183-8

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