Total chemical synthesis of a ribozyme derived from a group I intron.

Abstract

We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The (19). Here, we report the chemical synthesis of these RNA fragments, procedures for their purification, and their assembly to form an active ribozyme. We also describe derivatives of these fragments with ribonucleotide analog substitutions at critical positions within the catalytic core and the effects of these substitutions on the activity and stability of the corresponding ribozymes.The chemical synthesis of RNA has several advantages over synthesis by transcription, primarily the potential for "atomic mutagenesis" in which individual atoms or chemical groups are altered or removed by the site-specific incorporation of nucleotide analogs. Advances in the solid-phase chemical synthesis of RNA (1) have allowed the complete chemical synthesis of small ribozymes, including 19-and 35-nt hammerhead ribozymes (1, 2) and a 59-nt hairpin ribozyme (3). The roles of individual 2'-hydroxyl groups, exocyclic amino groups, and N-7s in the hammerhead ribozyme have been examined by the synthesis of RNAs containing deoxynucleotides (1, 4), purine riboside (5), inosine (6), and 7-deazaadenosine (7). Other synthetic RNAs have also been reported in which analogs such as 2-aminopurine (8), inosine (9), and 2'-fluoro (10, 11), 2'-amino (12, 13), and 2'-O-methyl nucleotides (14) have been incorporated. The largest chemically synthesized RNA that has been reported is the 77-nt tRNAMet; however, its methionine-acceptance activity was only 11%. Incompletely deprotected species may have contributed to this low biological activity (15).The group I and group II self-splicing introns and the M RNA of RNase P are larger ribozymes, typically >200 nt long. These ribozymes are beyond the reach of current synthetic methods and have generally been synthesized by the transcription of DNA templates by T7 RNA polymerase. However, certain intrinsic properties of T7 RNA polymerase are undesirable (16) including (i) a strongly preferred 5' sequence, (ii) the addition of nontemplated bases at the 3' end of the transcript, and (iii) the inability of the polymerase to incorporate ribonucleoside analogs into specified positions in a sequence [although this last limitation has been partially relieved by the methods described by Moore and Sharp (17) (Qiagen, Chatsworth, CA.). The column was prewashed with 10 ml of 50 mM triethylammonium bicarbonate (pH 7.0) and the sample was loaded. After a 10-ml wash with 50 mM triethylammonium bicarbonate (pH 7.0), the RNA was eluted with 2 M triethylammonium bicarbonate (pH 7.5) and dried repeatedly.HPLC Purification. After deprotection, the RNAs were purified by gel electrophoresis on a 6% polyacrylamide/8 M urea gel. UV shadowing was used to visualize a major band of the expected size. Depending upon how much material was tPresent add...

FTIR Spectroscopic Studies of Oligonucleotides That Model a Triple-Helical Domain in Self-Splicing Group I Introns

“…In the proposed model, the G451 is involved, both in Watson-Crick base pairing to form the P6 helix and nucleoside triple interactions with J3/4, whereas U453 is involved in a nucleoside triple interaction. The absence of complexation in these cases could indicate either direct or indirect participation of 2′-OH groups at these positions (Chastain & Tinoco, 1992Whoriskey et al, 1995). Indirect participation would mean that the absence of 2′-OH group could result in a different sugar conformation.…”

A Synthetic Model for Triple-Helical Domains in Self-Splicing Group I Introns Studied by Ultraviolet and Circular Dichroism Spectroscopy

“…This would imply that the presence of the 2′-OH group in the ribose sugar might be partially involved in hydrogen bonding to form the required nucleoside triples for the association [cf. Whoriskey et al (1995) and Tinoco (1992a, 1993)]. Further work to probe this aspect is in progress and will be communicated separately.…”

Mutant ATP-binding RNA aptamers reveal the structural basis for ligand binding

“…Mutant aptamers containing 2 H deoxynucleotide substitutions were chemically synthesized on a Milligen Cyclone Plus DNA synthesizer using ribonucleoside phosphoramidites (Chemgenes) and deoxyribonucleoside phosphoramidites (Chemgenes) at the desired positions. For the single deoxyribose substitutions, each aptamer was synthesized as two separate fragments, a 23-nucleotide oligonucleotide with the internal loop and an 11-nucleotide oligonucleotide containing the bulged G. The RNAs and RNA-DNA hybrids were deprotected as described (Whoriskey et al, 1995) and gel puri®ed. The position of the 2 H deoxynucleotide substitution was con®rmed by partial alkaline hydrolysis of the oligonucleotides followed by gel electrophoresis.…”