Adjusting the Structure of 2′-Modified Nucleosides and Oligonucleotides via C4′-α-F or C4′-α-OMe Substitution: Synthesis and Conformational Analysis

Abstract

We report the first syntheses of three nucleoside analogues, namely, 2',4'-diOMe-rU, 2'-OMe,4'-F-rU, and 2'-F,4'-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4'-α-position of a 4',5'-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2',4'-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4'-α-F/OMe substituents reinforce the C3'-endo ( north) conformation of 2'-OMe-rU. Furthermore, the predominant C2'-endo ( south/ east) conformation of 2'-F-araU switches to C3'-endo upon introduction of these substituents at C4'. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4'-substituents can modulate the binding affinity of the parent 2'-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics.

Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity

“…Other than 2′F-RNA, and to a small degree 2′,5′-RNA, none of these modifications supported activity. This was surprising given the RNA-like (C3′- endo ) sugar pucker previously reported for 2′,4′-di-Cα- O Me and 2′F-4′-Cα- O Me (48,50,51). Multiple 4′- O -methyl groups may introduce unfavorable steric constraints due to their bulkiness relative to a hydrogen atom.…”

“…We used both commercially available and custom modified nucleotides that can mimic RNA or DNA nucleotides and better control crRNA conformation, flexibility, polar contacts and steric constraints. Among the chemically modified nucleotides we tested, those possessing conformational properties similar to RNA were 2′F-RNA, 2′,5′-RNA (31–33), LNA (44), 2′,4′-di-Cα- O Me RNA (51), 2′F-4′-Cα- O Me RNA (39,40) and 2′- O -methyl (57). Among these, 2′F-RNA is one of the most prominent RNA substitutions in nucleic acid-based applications because of its C3′- endo sugar pucker that increases binding affinity to target RNA, increased endonuclease resistance, and small substituent size (38,61,62).…”

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

“…In summary, crystallographic data for RNAs with Ufo, Ufob, Ufme, uo or uob residues provide insight into the stability, nuclease resistance and activity of siRNAs containing 4′- C -modified residues. Notes added in proof: Prior to acceptance of the revised version of this manuscript, a paper reporting the synthesis of uo and its incorporation into siRNAs appeared on-line ( 40 ).…”

2’-O-methylation alters the RNA secondary structural ensemble

“…In isolated mononucleotides and unpaired pyrimidine nucleotides, the ribose moiety exists in a 60:40 dynamic equilibrium between C3′-endo and C2′-endo sugar pucker conformations (32), whereas most paired nucleotides or nucleotides that stack intra-helically predominantly adopt the C3′-endo conformation (33). In pyrimidine nucleosides (Um and Cm) and dinucleotides (UmpU and CmpC), 2′-O-methylation biases the sugar pucker equilibrium in favor of C3′-endo by 0.1-0.6 kcal/mol ( Figure 1B) (34)(35)(36)(37). This bias arises due to intra-residue steric repulsion between the 2′-O-methyl, the 3′-phosphate, and the 2-carbonyl groups when in the C2′-endo conformation (34).…”