Interplay of Structure, Hydration and Thermal Stability in Formacetal Modified Oligonucleotides: RNA May Tolerate Nonionic Modifications Better than DNA

Abstract

DNA and RNA oligonucleotides having formacetal internucleoside linkages between uridine and adenosine nucleosides have been prepared and studied using UV thermal melting, osmotic stress and X-ray crystallography. Formacetal modifications have remarkably different effect on double helical RNA and DNA -the formacetal stabilizes RNA helix by +0.7 °C, but destabilizes DNA helix by -1.6 °C per modification. The apparently hydrophobic formacetal has little effect on hydration of RNA but decreases the hydration of DNA, which suggests that at least part of the difference in thermal stability may be related to differences in hydration. A crystal structure of modified DNA shows that two isolated formacetal linkages fit almost perfectly in an A-type helix (decamer). Taken together, the data suggest that RNA may tolerate nonionic backbone modifications better than DNA. Overall, formacetal appears to be an excellent mimic of phosphate linkage in RNA and an interesting modification for potential applications in fundamental studies and RNA based gene control strategies, such as RNA interference.The potential of RNA interference (RNAi) to become a new therapeutic approach stimulates interest in chemical modifications to optimize the efficacy of RNA-based drugs. 1 Several modifications, developed earlier to improve the properties of antisense oligonucleotides, have also shown promising results in RNAi.1 , 2 We are interested in developing RNA analogues having non-ionic internucleoside linkages.3 ,4 Among such, we have found that replacement of selected phosphates with amide linkages (CH 2 -CO-NH-5′) does not change the overall conformation and thermal stability of RNA. 3 Recently, Iwase et al. 5 reported that introduction of two such amide linkages at the overhanging uridines of an siRNA did not significantly change the RNAi activity. The formacetal linkage 3′-O-CH 2 -O-5′ is another interesting modification that has served as a phosphate mimic in structural studies on a photolyase bound Correspondence to: Eriks Rozners, erozners@binghamton.edu. Supporting Information Available: Experimental procedures, details of thermal melting and osmotic stress experiments, and copies of 1 H and 13 C NMR data. This material is available free of charge via the Internet at http://pubs.acs.org. Previous studies suggested that formacetal modification might have different effect on DNA and RNA oligonucleotides. Van Boom and co-workers found that formacetal modifications strongly destabilized DNA duplexes by -1.4 to -2.4 °C per modification.7 Gao and co-workers reported similar findings (-3 °C per modification), however, their preliminary NMR studies suggested that formacetal linkage caused little structural perturbation of DNA helix.8 Matteucci found that formacetals in the DNA strand only slightly decreased the stability of DNA-RNA heteroduplexes.9 In contrast, our preliminary studies showed that formacetals were somewhat stabilizing in all RNA duplexes (+0.2 to +0.8 °C per modification).4 However, each of these studies used different mo...

Amides as Excellent Mimics of Phosphate Linkages in RNA

“…Backbone-modified RNA may find broad application in the fundamental biology and biomedicine of noncoding RNAs, providing that the modifications mimic the structure of the phosphodiester linkage and do not alter the conformation of RNA. In particular, the potential of RNA interference to become a new therapeutic strategy has revitalized interest in chemical modifications that may optimize the pharmacological properties of short interfering RNAs (siRNAs).[1] We are interested in hydrophobic nonionic mimics of the phosphate backbone, such as formacetals [2] and amides, [3] that may confer high nuclease resistance to siRNAs along with reduced charge and increased hydrophobicity. Earlier studies showed that 3'-CH 2 -CO-NH-5' internucleoside amide linkages (abbreviated here as AM1) were well-tolerated in the DNA strand of an A-type DNA-RNA heteroduplex.…”

Primitive Genetic Polymers

“…Phosphorus (V) esters and acetals are both tetrahedral and approximately isosteric, and it is known that aNAs can base pair with RNAs (Matteucci and Bischofberger 1991). Specifically, point substitutions of this linkage impart a modest destabilization to DNA-DNA and DNA-RNA duplexes and show saltdependent effects on RNA-RNA duplexes (stabilization at low salt, destabilization at high salt) (Jones et al 1993;Rice and Gao 1997;Rozners et al 2007;Kolarovic et al 2009). …”

The many twists and turns of DNA: template, telomere, tool, and target

“…1.5°C per modification. In the crystal structure of an A-form DNA single formacetal modifications per strand fit seamlessly into the duplex, but exhibited poor hydration relative to phosphate groups [70•] (3hr3). Osmotic stressing studies using several small molecule cosolutes demonstrated that more water was released from formacetal-modified RNAs upon melting than from modified DNAs.…”