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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GGG-J (10))
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Fabian, Miles
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University of Texas Sw Medical Center Dallas
Schools of Medicine
United States
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Matsui, Masayuki; Corey, David R (2017) Non-coding RNAs as drug targets. Nat Rev Drug Discov 16:167-179
Matsui, Masayuki; Prakash, Thazha P; Corey, David R (2016) Argonaute 2-dependent Regulation of Gene Expression by Single-stranded miRNA Mimics. Mol Ther 24:946-55
Li, Liande; Matsui, Masayuki; Corey, David R (2016) Activating frataxin expression by repeat-targeted nucleic acids. Nat Commun 7:10606
Hu, Jiaxin; Liu, Jing; Li, Liande et al. (2015) Engineering Duplex RNAs for Challenging Targets: Recognition of GGGGCC/CCCCGG Repeats at the ALS/FTD C9orf72 Locus. Chem Biol 22:1505-1511
Chu, Yongjun; Wang, Tao; Dodd, David et al. (2015) Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells. Nucleic Acids Res 43:e75
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
Matsui, Masayuki; Threlfall, Richard N; Caruthers, Marvin H et al. (2014) Effect of 2'-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells. Artif DNA PNA XNA 5:e1146391
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
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; Prakash, Thazha P; Corey, David R (2013) Transcriptional silencing by single-stranded RNAs targeting a noncoding RNA that overlaps a gene promoter. ACS Chem Biol 8:122-6

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