Warning: Undefined array key "mm" in /www/wwwroot/www.ai-bt.com/si.php on line 10 Deprecated: trim(): Passing null to parameter #1 ($string) of type string is deprecated in /www/wwwroot/www.ai-bt.com/si.php on line 10 Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Literature DB >> 7544462

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

F Wincott¹, A DiRenzo, C Shaffer, S Grimm, D Tracz, C Workman, D Sweedler, C Gonzalez, S Scaringe, N Usman.

Abstract

Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.

Entities: Chemical

Mesh：

Substances：

Year: 1995 PMID： 7544462 PMCID： PMC307092 DOI： 10.1093/nar/23.14.2677

Source DB: PubMed Journal: Nucleic Acids Res ISSN： 0305-1048 Impact factor: 16.971

13 in total

Review 1. Exploiting the chemical synthesis of RNA.

Authors: N Usman; R Cedergren
Journal: Trends Biochem Sci Date: 1992-09 Impact factor: 13.807

2. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

Authors: S A Scaringe; C Francklyn; N Usman
Journal: Nucleic Acids Res Date: 1990-09-25 Impact factor: 16.971

3. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding.

Authors: M Koizumi; E Ohtsuka
Journal: Biochemistry Date: 1991-05-28 Impact factor: 3.162

4. The role of a conserved guanosine residue in the hammerhead-type RNA enzyme.

Authors: O Odai; H Hiroaki; T Sakata; T Tanaka; S Uesugi
Journal: FEBS Lett Date: 1990-07-02 Impact factor: 4.124

5. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

Authors: G Slim; M J Gait
Journal: Nucleic Acids Res Date: 1991-03-25 Impact factor: 16.971

6. Solid phase synthesis of the 5'-half of the initiator t-RNA from B. subtilis.

Authors: C Chaix; A M Duplaa; D Molko; R Téoule
Journal: Nucleic Acids Res Date: 1989-09-25 Impact factor: 16.971

7. Chemical synthesis of a biologically active natural tRNA with its minor bases.

Authors: D Gasparutto; T Livache; H Bazin; A M Duplaa; A Guy; A Khorlin; D Molko; A Roget; R Téoule
Journal: Nucleic Acids Res Date: 1992-10-11 Impact factor: 16.971

8. Effect of excess water on the desilylation of oligoribonucleotides using tetrabutylammonium fluoride.

Authors: R I Hogrefe; A P McCaffrey; L U Borozdina; E S McCampbell; M M Vaghefi
Journal: Nucleic Acids Res Date: 1993-10-11 Impact factor: 16.971

9. Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF).

Authors: E Westman; R Strömberg
Journal: Nucleic Acids Res Date: 1994-06-25 Impact factor: 16.971

10. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding.

Authors: D J Fu; L W McLaughlin
Journal: Biochemistry Date: 1992-11-17 Impact factor: 3.162

127 in total

1. High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

Authors: T P Shields; E Mollova; L Ste Marie; M R Hansen; A Pardi
Journal: RNA Date: 1999-09 Impact factor: 4.942

2. Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.

Authors: R W Siegel; L Bellon; L Beigelman; C C Kao
Journal: J Virol Date: 1999-08 Impact factor: 5.103

Synthesis, deprotection, analysis and purification of RNA and ribozymes.

Review 1. Exploiting the chemical synthesis of RNA.

2. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

3. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding.

4. The role of a conserved guanosine residue in the hammerhead-type RNA enzyme.

5. Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

6. Solid phase synthesis of the 5'-half of the initiator t-RNA from B. subtilis.

7. Chemical synthesis of a biologically active natural tRNA with its minor bases.

8. Effect of excess water on the desilylation of oligoribonucleotides using tetrabutylammonium fluoride.

9. Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF).

10. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding.

1. High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

2. Use of DNA, RNA, and chimeric templates by a viral RNA-dependent RNA polymerase: evolutionary implications for the transition from the RNA to the DNA world.

3. RNA double cleavage by a hairpin-derived twin ribozyme.

Review 4. Nuclease-resistant synthetic ribozymes: developing a new class of therapeutics.

5. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization.

6. NMR structure of a ribosomal RNA hairpin containing a conserved CUCAA pentaloop.

7. Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation.

8. The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site.

9. The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA.

10. Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations.