Chemically modified nucleic acids (CNAs) function as potential antigene-, antisense-, or RNA interference (RNAi)-based drugs, as model systems for native DNA and RNA, as chemical probes in diagnostics and in high-throughput genomics and drug target validation, or the analysis of protein-nucleic acid interactions, and as tools for structure determination. This application is a continuation of our research directed at CNAs, with the long-term objectives to optimize their structure and activity for future applications as antisense oligonucleotide (AON) and small interfering RNA (siRNA) therapeutics, to devise an etiology of nucleic acid structure, and to determine the origins of substrate recognition by selected DNA- and RNA-processing enzymes. We propose four aims of broad biological significance in understanding the consequences of chemical modification for nucleic acid structure and stability and, by probing nucleic acid-protein interactions structurally and functionally by way of CNAs, the principles affording substrate recognition and processing by RNase H and A- and Y-class DNA polymerases.
Aim (1) focuses on investigations of the conformational features underlying the stability and efficacy of modifications assessed in connection with the discovery and development of the next generation of AON and siRNA therapeutics. This work will be carried out in collaboration with two world leaders in R&D of nucleic acid drugs, Alnylam Pharmaceuticals Inc. and Isis Pharmaceuticals, Inc.
In Aim (2) we will scrutinize the pairing and structure of glycol nucleic acid (GNA), the simplest artificial pairing system with a phosphate backbone found to cross-pair with RNA. We will also use neutron macromolecular crystallography (NMC) to delve deeper into aspects of nucleic acid structure that have eluded characterization using standard techniques, such as the orientations of water molecules and ribose 22-hydroxyl groups. Work in Aim (3) is directed at RNase H, an endonuclease that plays a key role in antisense applications by way of destroying the mRNA targeted by certain AONs. By way of 3D structural data for complexes with duplexes that are bound but not cleaved, we will probe features of nucleic acids central to recognition. The conformational range of the strand opposite RNA tolerated by the enzyme will be gauged with 3D structures of complexes with AON/RNA hybrids.
In Aim (4) we will address the recent hypothesis that certain DNA polymerases appear to rely more on shape than hydrogen bonding for accurate and efficient replication. Building on our recent structures of CNAs containing 2,4-difluorotoluene (F, an apolar T mimic) and complexes of F-modified templates with a trans-lesion (Y-class) DNA Pol, we will determine structures of ternary Pol-DNA-dNTP complexes containing F or dFTP of a replicative (A-class) DNA Pol, and correlate these data with activity data in the pre-steady- and steady-states. The main tool to be used is X-ray crystallography. Other approaches we will rely on to achieve our objectives are synthetic organic chemistry, biochemical and molecular biology tools as well as thermodynamics, kinetics and single-crystal NMC.

Public Health Relevance

A comprehensive structure-based program to analyze and improve RNA affinity, chemical stability and ultimately efficacy of chemically modified antisense and siRNA oligonucleotides with implications for drug discovery of nucleic acid therapeutics, to gain insight into self-pairing and cross-pairing with DNA and/or RNA of nucleic acid analogs analyzed in the context of an etiology of nucleic acid structure, and, by using chemically modified nucleotides, to investigate and thereby refute or confirm existing hypotheses regarding the chemical and structural bases for substrate recognition by the RNase H endonuclease and nucleotide insertion by A- and Y-class DNA polymerases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055237-16
Application #
8292045
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Preusch, Peter C
Project Start
1997-02-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
16
Fiscal Year
2012
Total Cost
$297,641
Indirect Cost
$106,846
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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
Kowal, Ewa A; Lad, Rahul R; Pallan, Pradeep S et al. (2013) Recognition of O6-benzyl-2'-deoxyguanosine by a perimidinone-derived synthetic nucleoside: a DNA interstrand stacking interaction. Nucleic Acids Res 41:7566-76
Seth, Punit P; Pallan, Pradeep S; Swayze, Eric E et al. (2013) Synthesis, duplex stabilization and structural properties of a fluorinated carbocyclic LNA analogue. Chembiochem 14:58-62
Ketkar, Amit; Zafar, Maroof K; Maddukuri, Leena et al. (2013) Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity. Chem Res Toxicol 26:221-32
Pallan, Pradeep S; Yu, Jinghua; Allerson, Charles R et al. (2012) Insights from crystal structures into the opposite effects on RNA affinity caused by the S- and R-6'-methyl backbone modifications of 3'-fluoro hexitol nucleic acid. Biochemistry 51:7-9
Egli, Martin (2012) The steric hypothesis for DNA replication and fluorine hydrogen bonding revisited in light of structural data. Acc Chem Res 45:1237-46
Egli, Martin; Pallan, Pradeep S; Allerson, Charles R et al. (2011) Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides. J Am Chem Soc 133:16642-9
Kowal, Ewa A; Ganguly, Manjori; Pallan, Pradeep S et al. (2011) Altering the electrostatic potential in the major groove: thermodynamic and structural characterization of 7-deaza-2'-deoxyadenosine:dT base pairing in DNA. J Phys Chem B 115:13925-34
Jiang, Xiaohua; Egli, Martin (2011) Use of chromophoric ligands to visually screen co-crystals of putative protein-nucleic acid complexes. Curr Protoc Nucleic Acid Chem Chapter 7:Unit 7.15.1-8
Pallan, Pradeep S; Greene, Emily M; Jicman, Paul Andrei et al. (2011) Unexpected origins of the enhanced pairing affinity of 2'-fluoro-modified RNA. Nucleic Acids Res 39:3482-95

Showing the most recent 10 out of 38 publications