Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs (original) (raw)
Related papers
Nucleic Acids Research, 2014
RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3´-CH 2 -CO-NH-5´amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5´-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.
Amides as Excellent Mimics of Phosphate Linkages in RNA
Angewandte Chemie International Edition, 2011
After the discovery that RNA can catalyze chemical reactions, the number and variety of non-coding RNAs and the important roles they play in biology has been growing steadily. Backbone modified RNA may find broad applications in fundamental biology and biomedicine of non-coding 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 (RNAi) 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 non-ionic mimics of the phosphate backbone, such as formacetals and amides , that may confer high nuclease resistance to siRNAs along with reduced charge and increased hydrophobicity. Earlier work 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. [4] Subsequently, we found that AM1 modifications did not change the thermal stability of RNA-RNA duplexes. Most importantly, Iwase and-co-workers recently showed that AM1 amides were well tolerated in the 3´-overhangs of siRNAs.
Chemistry – A European Journal, 2019
The success of RNA interference (RNAi) as a research tool and potential therapeutic approach has reinvigorated interest in chemical modifications of RNA. Replacement of the negatively charged phosphates with neutral amides may be expected to improve bioavailability and cellular uptake of small interfering RNAs (siRNAs) critical for in vivo applications. In this study, we introduced up to seven consecutive amide linkages at the 3´-end of the guide strand of an siRNA duplex. Modified guide strands having four consecutive amide linkages retained high RNAi activity when paired with a passenger strand having one amide modification between its first and second nucleosides at the 5´-end. Further increase in the number of modifications decreased the RNAi activity; however, siRNAs with six and seven amide linkages still showed useful target silencing. While an siRNA duplex having nine amide linkages retained some silencing activity, the partial reduction of the negative charge did not enable passive uptake in HeLa cells. Our results suggest that further chemical modifications, in addition to amide linkages, are needed to enable cellular uptake of siRNAs in the absence of transfection agents. An siRNA having four consecutive phosphates of its guide strand replaced by amide linkages showed high silencing activity when the passenger strand had an amide linkage between its first and second nucleosides. Further increase in amide modification decreased the RNAi activity; however, siRNAs having up to nine amide linkages retained some silencing activity at higher concentrations.
Synthesis and gene silencing properties of siRNAs containing terminal amide linkages
2014
The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3 end, carrying 5 -phosphate and 3 -hydroxyl groups (siRNAs). Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3 overhang recognition by the PAZ domain and the 5 -phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA) moieties at 5 , and at 3 positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.
ACS chemical biology, 2018
Potential in vivo applications of RNA interference (RNAi) require suppression of various off-target activities. Herein, we report that replacement of a single phosphate linkage between the first and second nucleosides of the passenger strand with an amide linkage almost completely abolished its undesired activity and restored the desired activity of guide strands that had been compromised by unfavorable amide modifications. Molecular modeling suggested that the observed effect was most likely due to suppressed loading of the amide-modified strand into Ago2 caused by inability of amide to adopt the conformation required for the backbone twist that docks the first nucleotide of the guide strand in the MID domain of Ago2. Eliminating off-target activity of the passenger strand will be important for improving therapeutic potential of RNAi.
Optimization of Automated Synthesis of Amide-Linked RNA
ACS Omega
The recent FDA approval of several antisense and siRNA drugs illustrates the utility of nucleic acid chemical modifications, but numerous challenges remain for generalized nucleic acid therapeutics, urging the exploration of new modification strategies. Replacing backbone phosphates with amides has shown promise for enhancing siRNA activity, specificity, and nuclease resistance; however, amide-linked RNA has not been fully explored due to lengthy and low yielding manual amide coupling procedures. We have addressed this by automating the assembly of amidelinked RNA using an Expedite 8909 nucleic acid synthesizer and optimizing solid-phase synthesis conditions to achieve 91−95% yields in just 5 min of coupling time. The optimized protocol allowed synthesis of a 21-nucleotidelong siRNA guide strand having six consecutive amide linkages at the 3′-end with an overall yield of ∼1%. Our results show that the steric hindrance caused by bulky 2′-O protecting groups and steric hindrance of the solid support are the key optimization variables for improving the amide couplings.
New opportunities for designing effective small interfering RNAs
Scientific Reports, 2019
Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Here...