Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry

Vinayak, Ravi; Anderson, Pamela J.; McCollum, Christie; Hampel, Arnold

doi:10.1093/nar/20.6.1265

Cited by 38 publications

(20 citation statements)

References 22 publications

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“…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).…”

mentioning

confidence: 99%

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

Whoriskey

Usman

Szostak

1995

Proc. Natl. Acad. Sci. U.S.A.

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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...

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mentioning

confidence: 99%

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

Whoriskey

Usman

Szostak

1995

Proc. Natl. Acad. Sci. U.S.A.

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“…An extra stable hairpin loop (5ЈC(UUCG)G3Ј) was introduced into these RNAs as a substitute for the wild type hairpin (5ЈGUU3Ј), since the introduction of the stable hairpin loop into a hairpin ribozyme was shown to raise the thermal stability. 32) Neither S53, S57 or S59 was active in the absence of E14, but S57 and S59 were cleaved in the presence of E14 at the same site as the wild type (E50-S1 complex). S53 without a linker was inactive even with a large excess of E14.…”

Section: Rational Design Of Allosteric Hairpin Ribozymementioning

confidence: 98%

Regulation of Ribozyme Activity with Short Oligonucleotides

Komatsu

2004

Biological & Pharmaceutical Bulletin

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Both hammerhead and hairpin ribozymes, which are well known as small catalytic RNAs, can catalyze either cleavage or ligation of RNA with sequence specificity. Although they have been applied to gene therapy and gene discovery, by cutting the mRNAs of target genes, these ribozymes normally do not have the ability to regulate their catalytic activities. Therefore, allosteric control has been applied to the ribozymes. Watson-Crick base pairing has superior ability in molecular recognition, and the sequence of nucleic acids makes diversity of molecules possible. Therefore, we have tried to construct a new ribozyme of which activity is regulated by a specific oligonucleotide. The allosteric ribozyme has potentials of not only regulation of activity but also a sensor for gene expression.

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“…Alternative methods utilizing more labile and, consequently, less stable and more expensive N-protective groups have been described (McBride et al, 1986;Schulof et al, 1987;Uzanski et al, 1989;Vu et al, 1990;Vinayak et al, 1992;Sinha et al, 1993;Theisen et al, 1993;Reddy et al, 1994;Chen et al, 2000;Ferreira et al, 2004;Ferreira and Morvan, 2005). Even with these, deprotection can still necessitate lengthy exposure to 29% aqueous ammonia solution, the standard hydrolytic reagent (Schulhof et al, 1987;Vu et al, 1990;Vinayak et al, 1992;Boal et al, 1996).…”

Section: Introduction Smentioning

confidence: 99%

“…Even with these, deprotection can still necessitate lengthy exposure to 29% aqueous ammonia solution, the standard hydrolytic reagent (Schulhof et al, 1987;Vu et al, 1990;Vinayak et al, 1992;Boal et al, 1996).…”

Section: Introduction Smentioning

confidence: 99%

“…These include variations on the theme of ammonia gas or ammonia solutions in ethanol or methylamine (Vinayak et al, 1992;Reddy et al, 1994Reddy et al, , 1995Boal et al, 1996;Iyer et al, 1997), alkali metal hydroxide-based reagents (Köster et al, 1981;Surzhikov et al, 2000;Kumar et al, 2002;Kumar and Gupta, 2003), or even more complex mixtures, such as hydrazine:ethanolamine:methanol (Polushin et al, 1994). However, many biological and biomedical applications are compromised by the presence of metal ions, which some of these reagents would incur.…”

Section: Introduction Smentioning

confidence: 99%

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Fast Deprotection of Synthetic Oligodeoxyribonucleotides Using Standard Reagents under Microwave Irradiation

et al. 2008

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Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dABz , dC Bz , dC Ac , dG ibu , dG PAC ) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA

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Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry

Cited by 38 publications

References 22 publications

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

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

Regulation of Ribozyme Activity with Short Oligonucleotides

Fast Deprotection of Synthetic Oligodeoxyribonucleotides Using Standard Reagents under Microwave Irradiation

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