5′-C-Malonyl RNA: Small Interfering RNAs Modified with 5′-Monophosphate Bioisostere Demonstrate Gene Silencing Activity

Abstract

5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.

Squaramide‐Based 5’‐Phosphate Replacements Bind to the DNA Repair Exonuclease SNM1A

“…Mononucleotide analogues with isosteres at the 5’‐position are being investigated as antiviral agents, as the introduction of the mimic overcomes the rate limiting phosphorylation of the nucleoside drug . The activity of siRNA depends on 5’‐terminal phosphorylation by kinases, which limits chemical modifications of siRNA as extensive modification can compromise recognition by kinases . The introduction of bioisosteres could circumvent this issue.…”

“…Substitution of the phosphate group by 5’‐methylenephosphonate lowered the activity of single stranded siRNA, but conserved it for double stranded siRNA . A 5’‐malonate group retained or enhanced silencing activity and metabolic stability of siRNA compared to its phosphorylated analogue . However, the use of terminal 5’‐phosphate replacements on oligonucleotides is challenging, as the mimetics need to withstand solid‐phase oligonucleotide synthesis or be amenable to introduction using mild conditions post solid‐phase synthesis.…”

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

“…[4] Examples are manifold and include nucleobase-, ribose-,a nd backbone modifications that, for instance, can improve pharmacokinetic properties of therapeutic oligonucleotides. [5,6] Other examples include isoptope-and dye-labeled RNAs for structural and functional analysisu sing NMR andf luorescence spectroscopy. [7][8][9] Solid-phase synthesis using nucleoside phosphoramidites is the method of choice to introduce am odificationo fi nterest in as ite-specific manner into RNA.…”

“…Likewise, artificial modifications have become increasingly important for the manipulation of nucleic acid properties and to create chemical diversity that is needed for applications in the life sciences . Examples are manifold and include nucleobase‐, ribose‐, and backbone modifications that, for instance, can improve pharmacokinetic properties of therapeutic oligonucleotides . Other examples include isoptope‐ and dye‐labeled RNAs for structural and functional analysis using NMR and fluorescence spectroscopy …”

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

“…[1] Furthermore,s tructural modification of the 5'-end of therapeutic siRNAa nd mRNAi ncreases stability,e nhances uptake,a nd can modulate immunogenicity. [2] These 5'-modified RNAs are currently prepared by co-transcription, ligation, or de novo chemical synthesis utilizing chemically modified nucleotides.D irect chemical 5'-functionalization of native RNAi na queous solution, by reaction with the 5'phosphate,would be an attractive alternative;however,such methods have so far not been reported. Herein, we disclose the selective alkylation of the RNA5'-phosphate using diazo reagents,a nd its application in 5'-biotinylation and the chemical capping of an oligonucleotide.…”