Compounds that sequence-specifically bind target regions in chromosomal DNA in living cells hold tremendous potential as therapeutics, general research tools and diagnostic probes. DNA-targeting probes can modulate expression of genes involved in diseases, facilitate correction of detrimental mutations by targeted delivery of DNA-modifying agents or allow direct fluorescent labeling of chromosomal DNA. Progress in this area has been achieved with triplex forming oligonucleotides, minor groove binding polyamides, and peptide nucleic acids, but major methodological limitations have impeded development of drug candidates. Accordingly, there remains an extensive need for a DNA-targeting strategy which exhibits a) fast, high affinity binding, b) gene- specific targeting (>15 bp) of chromosomal DNA without sequence restrictions at physiologically relevant ionic strengths, c) excellent biostability, d) uncomplicated DNA-like handling and e) straightforward design. We propose Invader Locked Nucleic Acids (LNAs) as a solution to this grand challenge. This strategy is based on our discovery that: a) oligonucleotides modified with intercalator-functionalized 2'-amino-?-L-LNA monomers exhibit unsurpassed affinity toward DNA targets, b) duplex probes with 'monomer zippers'are exceedingly unstable, which c) generates a thermodynamic driving force for duplex probes to dissociate and bind double stranded DNA targets. The objective of this proposal is to maximize the DNA-targeting affinity of Invader LNA and to test the hypothesis that optimized Invader LNA can target chromosomal DNA and modulate gene expression in living cells. Specifically, we propose to a) optimize DNA-targeting of Invader LNA by chemical structural engineering of the monomer zippers (energetic hotspots) and the probe architecture, and b) demonstrate binding and modulation of gene expression by optimized Invader LNA in specific gene promoter regions using cultured cell models - these gene promoter regions will be studied in co-transfected plasmids and in the context of endogenous chromosomal DNA. The proposed experiments will clearly establish the feasibility of Invader LNAs as a paradigm changing DNA-targeting strategy. The successful development of Invader LNA as a reliable, biostable and easily handled probe technology will allow: a) strong, sequence- unrestricted and site-specific binding to accessible target regions of dsDNA under physiological conditions, b) modulation of gene expression, and/or c) targeted delivery of functional entities (DNA-modifying agents, donor DNA, chain cutters, optical labels or drugs) and will result in a marked acceleration in the development of drug candidates and tools for site-specific manipulation of DNA and have far-reaching implications in medicine, biology, and biotechnology.

Public Health Relevance

The availability of oligonucleotide-based probes, which a) allow reliable and site-specific binding under physiological conditions to accessible target regions of any sequence in plasmid or chromosomal DNA and b) facilitate efficient modulation of gene expression, will herald a new era in the treatment of gene-based pathologies. Every disease or malady that is the result of mutation or dis-regulated gene expression may be treatable by carefully designed and targeted invader LNAs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088697-03
Application #
8115025
Study Section
Special Emphasis Panel (ZGM1-CBB-7 (EU))
Program Officer
Sledjeski, Darren D
Project Start
2009-08-01
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
3
Fiscal Year
2011
Total Cost
$171,914
Indirect Cost
Name
University of Idaho
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
075746271
City
Moscow
State
ID
Country
United States
Zip Code
83844
Karmakar, Saswata; Guenther, Dale C; Gibbons, Bradley C et al. (2017) Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2'-triphenylene- and coronene-functionalized RNA monomers. Org Biomol Chem 15:9362-9371
Hrdlicka, Patrick J; Karmakar, Saswata (2017) 25 years and still going strong: 2'-O-(pyren-1-yl)methylribonucleotides - versatile building blocks for applications in molecular biology, diagnostics and materials science. Org Biomol Chem 15:9760-9774
Anderson, Brooke A; Hrdlicka, Patrick J (2016) Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes. J Org Chem 81:3335-46
Anderson, Brooke A; Onley, Jared J; Hrdlicka, Patrick J (2015) Recognition of Double-Stranded DNA Using Energetically Activated Duplexes Modified with N2'-Pyrene-, Perylene-, or Coronene-Functionalized 2'-N-Methyl-2'-amino-DNA Monomers. J Org Chem 80:5395-406
Anderson, Brooke A; Karmakar, Saswata; Hrdlicka, Patrick J (2015) Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes. Molecules 20:13780-93
Guenther, Dale C; Karmakar, Saswata; Hrdlicka, Patrick J (2015) Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles. Chem Commun (Camb) 51:15051-4
Anderson, Brooke A; Hrdlicka, Patrick J (2015) Synthesis and characterization of oligodeoxyribonucleotides modified with 2'-thio-2'-deoxy-2'-S-(pyren-1-yl)methyluridine. Bioorg Med Chem Lett 25:3999-4004
Guenther, Dale C; Anderson, Grace H; Karmakar, Saswata et al. (2015) Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications. Chem Sci 6:5006-5015
Kumar, Pawan; Baral, Bharat; Anderson, Brooke A et al. (2014) C5-alkynyl-functionalized ?-L-LNA: synthesis, thermal denaturation experiments and enzymatic stability. J Org Chem 79:5062-73
Guenther, Dale C; Kumar, Pawan; Anderson, Brooke A et al. (2014) C5-amino acid functionalized LNA: positively poised for antisense applications. Chem Commun (Camb) 50:9007-9

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