Structural Studies and Gene Silencing Activity of siRNAs Containing Cationic Phosphoramidate Linkages

Address correspondence to: Masad J. Damha, PhD, Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, Quebec H3A 0B8, Canada, E-mail: [email protected]

Introduction

Oligonucleotide analogues have tremendous potential for use in a wide variety of applications, including diagnostics and therapeutics. Short interfering RNAs (siRNAs) can be used to regulate gene expression through the RNA interference (RNAi) pathway. These 21mer double-stranded RNA molecules induce the degradation of complementary sequences of target messenger RNA, preventing translation and thus effecting knockdown of specific genes [1].

Chemical modifications of the sugar, nucleobase, and internucleotide linkage of oligonucleotides have been used to address many of the undesirable properties of oligonucleotides and to accelerate their clinical development [2-5]. In particular, modified internucleotide linkages have been extensively studied; some notable examples include phosphorothioates [6], methylphosphonates [7], boranophosphates [8], and amides [9]. Such backbone modifications have been used to alter nuclease resistance [8,10,11], biological activity [12], duplex thermal stability [10,12-14], and cellular uptake [15,16], leading to the development of a number of oligonucleotide therapeutics in clinical trials.

Phosphoramidate (PN) modifications of oligonucleotides, in which one of the non-bridging oxygen atoms of the phosphate linkage is replaced by an amino moiety (Fig. 1), were first reported in 1986 [17]. Since then, these modifications have been explored in the context of both antigene [18-20] and antisense [21-24] approaches; in each of these strategies, the active agent is a single strand of DNA [25]. In general, PN-modified oligonucleotides exhibit improved nuclease resistance relative to unmodified DNA; in addition, the introduction of cationic linkages within the oligonucleotide backbone could potentially facilitate cellular uptake due to the reduction of the net negative charge of the phosphodiester backbone [25]. Other biological properties of PNs vary depending on the structure and biophysical properties of the amine conjugate [26]; as the incorporation of PN linkages creates chirality at the phosphorus center, the stereochemistry of the phosphorus atom also plays a role [27,28]. Cationic alkylamine conjugates have been widely used in the exploration of PN oligonucleotides [19,27,29-31].

FIG. 1. General structures of phosphodiester (left) and non-bridging phosphoramidate (right) linkages. Color images...