Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.

Small interfering double-stranded RNAs have been synthesized bearing one or more base modifications at nucleotide positions 4, 11 and/or 16 in the guide strand. The chemically modified base is an N2-alkyl-8-oxo-7,8-dihydroguanine (alkyl = propyl, benzyl) that can alternatively pair in a Watson-Crick sense opposite cytosine (C) or as a Hoogsteen pair opposite adenine (A). Cellular delivery with C opposite led to effective targeting of A-containing but not C-containing mRNA sequences in a dual luciferase assay with RNA interference levels that were generally as good as or better than unmodified sequences. The higher activity is ascribed to an inhibitory effect of the alkyl group projecting into the minor groove of double-stranded RNA preventing off-target binding to proteins such as PKR (RNA-activated protein kinase).

N 2 -Alkyl analogues of 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N 2 -alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-protein interactions in the minor vs. major grooves.Chemical modification of nucleosides has been a successful strategy for antiviral, 1 antimetabolite, 2 antitumor, 3,4 and diagnostic agents. 4 Modified nucleosides in oligomers exist naturally due to cellular reactions on both the bases and the sugars of DNA and RNA and have also been introduced into oligomers synthetically. Modified nucleosides are common in the RNA field due to applications in investigating reaction mechanisms, 5,6 imparting favorable properties on siRNAs, 7,8 probing RNA structure and function, 9-11 and exploring interactions between RNA and proteins or small molecules. 1 Both sugar and backbone modifications have been explored to improve nuclease stability and target identification in antisense and siRNA approaches; 12,13 however, examples of base modification in these applications are few by comparison. [14][15][16] The ability to alter the hydrophobicity and steric properties of the major and minor groove appeared to us as a possible means of modulating both interstrand interactions as well as nucleic acid-protein interactions 7,17,18 thereby expanding the range of nucleic acid modifications in the design of DNA and RNA therapeutics.Inspiration for the present work came from one of the major products of oxidative damage to the DNA, namely 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG). 19- group at C8 of the purine increases the propensity of the purine to flip from the normal anti conformation to syn, where it exposes the Hoogsteen face of the purine to base pairing. Like its parent guanine, OG(anti) accepts cytosine as a Watson-Crick partner, while the complementary base for OG(syn) is adenosine ( Figure 1). During the anti-syn conformational change, the N 2 -amino group and the C8-oxo groups exchange positions between the minor and major grooves ( Figure 1). As a result, addition of an alkyl or aryl group to the exocyclic amine should enable the placement of the substituent in either groove, in a way that will be governed by the identity of the base opposite.OG can be synthetically incorporated into DNA and RNA oligomers via the corresponding phosphoramidite. While we, among others, have been studying OG and its incorporation into oligomers, including chemical characterization and enzymology with DNA processing enzymes, 22,23 OG is rarely studied in RNA. Thermal denaturation studies, 22,24,25 NMR studies, 26 and x-ray crystallographic structures 23,27,28 of the OG(syn):A(anti) base pair show that the B-form DNA helix is only very slightly perturb...

Exposure of cells to phenolic compounds through exogenous and endogenous sources can lead to deleterious effects via nucleobase modifications of DNA occurring under oxidative conditions. 2'-Deoxyguanosine (dG) is the most electron rich of the four canonical bases and includes many nucleophilic sites; it is also susceptible to oxidation with numerous reactive oxygen species. In these studies, dG was allowed to react with 2-naphthol in the presence of copper or iron salts yielding two principal isomeric products. Spectroscopic analysis and reactions with alkylated nucleosides support the assignment of compound 1a/1b as a pair of atropisomer N(2) adducts and compound 2a/2b as a diastereomeric mixture of tricyclic [4.3.3.0] adducts. Both products are the result of an overall four-electron oxidation process and consequently have the same masses, though drastically different structures, providing mechanistic insight into their formation. Thus, dG alkylation by 2-naphthol under oxidative conditions yields products whose structural properties are altered, leading to potentially mutagenic effects in genomic DNA.