Site-Selective RNA Functionalization via DNA-Induced Structure.

Methods for RNA functionalization at specific sites are in high demand but remain a challenge, particularly for RNAs produced by transcription rather than by total synthesis. Recent studies have described acylimidazole reagents that react in high yields at 2'-OH groups stochastically at nonbase-paired regions, covering much of the RNA in scattered acyl esters. Localized reactions, if possible, could prove useful in many applications, providing functional handles at specific sites and sequences of the biopolymer.

Effect of 2'-5'/3'-5' phosphodiester linkage heterogeneity on RNA interference.

We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2.

Reversible RNA acylation for control of CRISPR-Cas9 gene editing.

We report the development of post-transcriptional chemical methods that enable control over CRISPR-Cas9 gene editing activity both in assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated ("cloaked") sgRNA was completely inactive in DNA cleavage with Cas9 , and activity was quantitatively restored after phosphine treatment.

Polyacetate and Polycarbonate RNA: Acylating Reagents and Properties.

Acylation of RNA at 2'-OH groups is widely applied in mapping RNA structure and recently for controlling RNA function. Reactions are described that install the smallest 2-carbon acyl groups on RNA-namely, 2'-O-acetyl and 2'-O-carbonate groups. Hybridization and thermal melting experiments are performed to assess the effects of the acyl groups on duplex formation.

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

CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis.

Structural properties and gene-silencing activity of chemically modified DNA-RNA hybrids with parallel orientation.

We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reverse Watson-Crick base pairs in ps-DNA:DNA, ps-DNA:RNA, ps-(DNA-2'F-ANA):RNA, and ps-DNA:2'F-RNA duplexes. The formation of these duplexes was confirmed by UV melting experiments, FRET and CD studies.

Seven-Membered Ring Nucleoside Analogues: Stereoselective Synthesis and Studies on Their Conformational Properties.

The synthesis of a novel series of seven-membered ring nucleoside analogues as candidates for biological screening and gene silencing applications is described. The key step in the synthetic approach is a stereoselective synthesis of an epoxide that is used as a common synthetic intermediate to prepare functionalized oxepane nucleoside derivatives. The conformational landscape and preferred ring-puckering of selected oxepane nucleosides was also studied by NMR, X-ray crystallography, and quantum mechanical calculations.

Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT.

A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT.