Discovery of RNA interference (RNAi) has reinvigorated interest in chemical modifications to optimize properties of small interfering RNAs (siRNAs). The long-term goal of our research is to explore RNA's structure and function using chemical approaches and to develop modifications for practical application in RNAi. The present proposal focuses on internucleoside amides as non-ionic mimics of the phosphodiester linkages and will test the hypotheses that amides (1) can be readily introduced in RNA using solid-phase synthesis, (2) are excellent mimics of the phosphate backbone of RNA, and (3) will increase enzymatic stability and cellular uptake of siRNAs without compromising their RNAi activity. We also envision that amides may improve biodistribution and pharmacokinetics of siRNAs. We propose an interdisciplinary (organic chemistry, structural biochemistry and RNA biology) study with the specific aims to: 1. Develop synthetic methods to introduce consecutive amide linkages at any desired location in RNA by adopting and optimizing solid-phase peptide, PNA, and RNA synthesis methods. 2. Confirm that amide-linked RNA can mimic the structure of natural RNA using UV spectroscopic and X- ray crystallographic techniques in collaboration with Prof. Martin Egli (Vanderbilt University). 3. Synthesize siRNAs having several amide linkages at the 3'-end of each strand and test their biological properties and RNAi activity in collaboration with Dr. Devin Leake (Dharmacon). Amides may offer several advantages for in vivo RNAi applications: (1) high nuclease resistance due to the absence of the natural phosphate; (2) enhanced cellular uptake due to the reduction of the negative charge; (3) improved biodistribution and pharmacokinetics due to the increased hydrophobicity. Despite these potentially beneficial properties, neither amides nor any other non-ionic linkages have been tested in RNAi. If accepted by RNAi proteins, amides may significantly improve properties of siRNAs and may be used to design a novel class of chemically modified siRNAs. Combination of synthetic chemistry, structural studies and RNA biology will provide unique insights into how chemical modifications (amides) influence conformation, hydration, and thermal stability of RNA. Such knowledge is important for rational design of nucleic acid analogues and for developing gene selective therapeutic agents for such long standing problems as cancer, viral infections, genetic disorders, and neurodegenerative diseases. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM071461-01A2
Application #
7178002
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Preusch, Peter C
Project Start
2007-03-01
Project End
2012-02-29
Budget Start
2007-03-01
Budget End
2008-02-29
Support Year
1
Fiscal Year
2007
Total Cost
$283,712
Indirect Cost
Name
Northeastern University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001423631
City
Boston
State
MA
Country
United States
Zip Code
02115
Hnedzko, Dziyana; McGee, Dennis W; Karamitas, Yannis A et al. (2017) Sequence-selective recognition of double-stranded RNA and enhanced cellular uptake of cationic nucleobase and backbone-modified peptide nucleic acids. RNA 23:58-69
Mutisya, Daniel; Hardcastle, Travis; Cheruiyot, Samwel K et al. (2017) Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs. Nucleic Acids Res 45:8142-8155
Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks (2016) Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye. Bioorg Med Chem 24:4199-4205
Anosova, Irina; Kowal, Ewa A; Sisco, Nicholas J et al. (2016) Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes. Chembiochem 17:1705-8
Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks (2014) Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids. Methods Mol Biol 1050:83-94
Mutisya, Daniel; Selvam, Chelliah; Lunstad, Benjamin D et al. (2014) Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs. Nucleic Acids Res 42:6542-51
Hnedzko, Dziyana; Cheruiyot, Samwel K; Rozners, Eriks (2014) Using triple-helix-forming Peptide nucleic acids for sequence-selective recognition of double-stranded RNA. Curr Protoc Nucleic Acid Chem 58:4.60.1-23
Tanui, Paul; Kennedy, Scott D; Lunstad, Benjamin D et al. (2014) Synthesis, biophysical studies and RNA interference activity of RNA having three consecutive amide linkages. Org Biomol Chem 12:1207-10
Muse, Oluwatoyosi; Zengeya, Thomas; Mwaura, Juddy et al. (2013) Sequence selective recognition of double-stranded RNA at physiologically relevant conditions using PNA-peptide conjugates. ACS Chem Biol 8:1683-6
Zengeya, Thomas; Gindin, Artem; Rozners, Eriks (2013) Improvement of sequence selectivity in triple helical recognition of RNA by phenylalanine-derived PNA. Artif DNA PNA XNA 4:69-76

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