Venkatraman Mohan

Oligonucleotide Conjugates: Alteration of the Pharmacokinetic Properties of Antisense Agents

Abstract Cholic acid, cholesterol, several polyamines and polyethylene glycols were conjugated to antisense oligonucleotides targeted to human or murine intercellular adhesion molecule-1 (ICAM-1) mRNA to study their effects on cellular absorption.

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR.

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds l-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 μM.

The Influence of Antisense Oligonucleotide-induced RNA Structure on Escherichia coli RNase H1 Activity

The ability of Escherichia coli RNase H1 to hydrolyze structured substrates containing antisense oligonucleotides preannealed to a 47-mer RNA was compared with its ability to hydrolyze unstructured substrates containing antisense oligonucleotides duplexed with 13-mer RNA. These results demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significantly slower than when the same oligonucleotides were bound to unstructured oligoribonucleotides. Structured substrates exhibited fewer cleavage sites, and each cleavage site was cleaved less rapidly than in unstructured substrates. Furthermore, the enzymatic activity ofE. coli RNase H1 for the structured substrates was most affected when the cleavage sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates. Molecular modeling suggests that the observed ablation of RNase H activity was due to the steric hindrance of the enzyme by the structured RNA, i.e. steric interference of the phosphate groups on the substrate and/or the binding site of the enzyme. When chimeric oligonucleotides composed of a five-base deoxynucleotide sequence flanked by chemically modified nucleotides were bound to structured RNA, the resultant duplexes were even worse substrates for RNase H. These results offer further insights into the role of antisense-induced RNA structure on RNase H activity and may facilitate the design of effective antisense oligonucleotides.

Molecular dynamics and NMR studies of single-stranded PNAs

Abstract Proton NMR spectroscopy and molecular dynamics simulations are employed to investigate the conformations of PNA monomers, a dimer and an octamer. The monomers exist as a 70:30 mixture of two amide rotamers interconverting slowly on the NMR time scale at 20 °C. In the major form, the side chain carbonyl group points toward the glycine, which places the methylene protons in proximity to the 2-aminoethyl protons. The minor form places its side chain carbonyl group away from the glycine, and the methylene protons are close in space to the glycine α protons. The PNA CT-dimer has multiple rotamers at 20 °C. In contrast, a NOESY spectrum taken from an octamer indicates only a single conformer in solution at 40 °C.

Solution phase combinatorial chemistry. Discovery of 13- and 15-membered polyazapyridinocyclophane libraries with antibacterial activity

Abstract A solution phase simultaneous addition of functionalities (SPSAF) combinatorial approach was utilized to synthesize 40 polyazacyclophane libraries (total complexity of 4275). Eighteen different functionality sets, utilizing 42 functionalities, were designed to disrupt RNA-protein interactions. Guanidine functionality sets with a greater potential to form positive charges provided the most active libraries. Differences in antibacterial activity are clearly related to different ring sizes with the more rigid 13-membered scaffold affording more active libraries compared with libraries from the 15-membered scaffold. Molecular modeling established a significant difference in the shapes of 13- and 15-membered pyridinophanes. Several libraries exhibited potent antibacterial activity.

Oligonucleotides bearing cationic groups: N2-(3-aminopropyl)deoxyguanosine. Synthesis, enhanced binding properties and conjugation chemistry

A phosphoramidite with an aminopropyl group placed at the N2- position of 2′-deoxyguonosine has been synthesized and incorporated into oligonucleotides. This modification shows enhanced binding properties against both DNA and RNA targets and is useful for conjugating other functionalities.

Conjugated Antisense Oligonucleotides

Abstract We have employed chemical modification strategies to improve cellular ahsorption of oligonucleotides. These include the conjugation of various pendant moieties to the oligonucleotide to affect its overall physical properties such as hydrophobicity, charge, and amphipathicity as well as pendants that may mediate absorption by binding to certain cellular receptors which internalize specific ligands. Our laboratory has prepared polyamines, polyethylene glycols and lipidic constituents conjugated to oligonucleotidcs in order to study their effects in enhancing absorption of antisense agents. These conjugates were targeted against human or murine Intercellular Adhesion Molecule- 1 (ICAM-1) mRNA.

2′-O-Carbamate-containing oligonucleotides: synthesis and properties

Abstract In order to evaluate the effect of a new 2′-carbohydrate modification on the hybridization properties of oligonucleotides, uridine 2′- O -carbamates were synthesized and incorporated into DNA strands. The key intermediate in the synthesis, a mixed succinimide carbonate 2 , was treated with various amines to give 2′- O -carbamates 3 . Thermal melting studies of modified oligonucleotides revealed that the presence of the 2′- O -carbamate modification significantly destabilized DNA/RNA duplexes. A molecular-modeling study indicated that unfavorable steric interactions between the hydrogen of the NH group from the carbamate substituent and the anomeric hydrogen of the sugar residue on the same strand of the duplex may be the contributing factor causing destabilization.

Structure and dynamics of MMI linked nucleotides

Abstract Results are presented on molecular dynamics (MD) simulations of (T∗T) dimers linked by a novel chemical functionality — the m ethylene m ethyl i mino (MMI) backbone designed as a neutral replacement for the negatively charged phosphodiester group in wild type nucleic acids. Simultaneous conformational transitions among the MMI backbone atoms during the course of the simulation have been observed, equivalent to inversion-rotation at the nitrogen atoms of the N-O bond. This process yields two families of low-energy conformations which maintain base stacking. The solution structure of the dimer has been studied by one and two-dimensional 1H NMR spectroscopy, and two slowly exchanging forms are observed at low temperature. Simulated NOESY spectra generated from the MD structures match the experimental NOESY data.

Vibrational Analysis of Phosphorothioate DNA: II. The POS Group in the Model Compound Dimethyl Phosphorothioate [(CH3O)2(POS)]-

The results of Raman and Infrared (IR) spectroscopic investigations on the vibrational modes of dimethyl phosphorothioate (DMPS) anion, [(CH3O)2(POS)]-, are reported. Ab initio calculations of the vibrational modes, the IR and Raman spectra and the interatomic force constants of DMPS were performed. A normal mode calculation was performed and the results were used to calculate the potential energy distribution for the vibrational modes. This analysis shows that in DMPS the P-S stretching mode has a frequency of about 630 cm-1 and an angle bending mode involving the sulfur atom has a frequency of about 440 cm-1. The proposed vibrational mode assignments will serve as marker bands in the conformational studies of phosphorothioate oligonucleotides which play a central role in the novel antisense therapeutic paradigm.