Design of hairpin ribozyme variants with improved activity for poorly processed substrates

Abstract

Application of ribozymes for knockdown of RNA targets requires the identification of suitable target sites according to the consensus sequence. For the hairpin ribozyme, this was originally defined as Y−2N−1*G+1U+2Y+3B+4, with Y = U or C, and B = U, C or G, and C being the preferred nucleobase at positions −2 and +4. In the context of development of ribozymes for destruction of an oncogenic mRNA, we have designed ribozyme variants that efficiently process RNA substrates at U−2G−1*G+1U+2A+3A+4 sites. Substrates with G−1*G+1U+2A+3 sites were previously shown to be processed by the wild‐type hairpin ribozyme. However, our study demonstrates that, in the specific sequence context of the substrate studied herein, compensatory base changes in the ribozyme improve activity for cleavage (eight‐fold) and ligation (100‐fold). In particular, we show that A+3 and A+4 are well tolerated if compensatory mutations are made at positions 6 and 7 of the ribozyme strand. Adenine at position +4 is neutralized by G6→U, owing to restoration of a Watson–Crick base pair in helix 1. In this ribozyme–substrate complex, adenine at position +3 is also tolerated, with a slightly decreased cleavage rate. Additional substitution of A7 with uracil doubled the cleavage rate and restored ligation, which was lost in variants with A7, C7 and G7. The ability to cleave, in conjunction with the inability to ligate RNA, makes these ribozyme variants particularly suitable candidates for RNA destruction.

Synthesis of Specifically Modified Oligonucleotides for Application in Structural and Functional Analysis of RNA

“…Thus, chemical synthesis of RNA is limited by the length of the oligomers. Up to now, we, therefore, preferred to synthesize nonmodified RNA molecules which are longer than 70 bases by in vitro transcription techniques as described [17]. Shorter nonmodified and modified RNAs in our laboratory are prepared by solid phase synthesis.…”

“…Shorter nonmodified and modified RNAs in our laboratory are prepared by solid phase synthesis. Our procedure is based on 2′- O - tert- butyldimethylsilyl (TBDMS) nucleoside protection and involves sequential couplings of β -cyanoethyl-( N,N ′-diisopropyl)phosphoramidites of 5′- O -dimethoxytrityl-2′- O -TBDMS nucleosides essentially by the method as described [17]. For assembly of the oligoribonucleotides, we predominantly use CPG or polystyrene beads to which the first ribonucleoside derivative is attached via its 3′-OH group as a succinate linkage.…”

“…All syntheses were carried out “trityl-off”. The obtained RNA was cleaved from the support and deprotected using NH 3 (30%) and ethanolic methylamine in a 1 : 1 mixture and TEAx3HF as described [17] and purified by gel electrophoresis using 10% denaturing polyacrylamide gels. Elution was carried out using 0.5 M LiOAc followed by EtOH precipitation.…”

Synthesis of a bifunctional cytidine derivative and its conjugation to RNA for in vitro selection of a cytidine deaminase ribozyme

“…The RNA GUC AGC CGU CAG GAU CCG UG used as model for studying 3'-terminal conjugation of 1 was synthesized on an Applied Biosystems 394 DNA/RNA Synthesizer following the standard protocol for oligoribonucleotide chain assembly. The synthesized RNA was deprotected using aqueous ammonia (32%)/aq methylamine (40%) (1:1, v/v) at 65 °C for 30 min for removal of base and phosphate protecting groups and cleavage from the support, and TEA·3HF for removal of 2'- O -protecting groups, essentially as described previously [43]. …”

Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

“…Hairpin ribozymes that form a stable structure, such that fragments remain bound, favor ligation, whereas hairpin ribozymes that are less stable, such that cleavage fragments can easily dissociate, favor cleavage (Fedor 1999, Welz et al 2003. These characteristic features distinguish the hairpin ribozyme from other small ribozymes, and we have shown in previous work that structural manipulation of hairpin ribozyme variants allows tuning of cleavage and ligation activity (Welz et al 2003;Ivanov et al 2005;Vauleon et al 2005;Drude et al 2007Drude et al , 2011Pieper et al 2007;Müller 2013, Balke et al 2014). Among these variants is a hairpin ribozyme that can cleave off its 5 ′ -and 3 ′ -end (Pieper et al 2007).…”