Nucleic Acids Research, 1990, Vol. 18, No. 18 5433-5441
© 1990
CHEMISTRY |
Chemical synthesis of biologically active oligoribonucleotides using ß-cyanoethyl protected ribonucleoside phosphoramidites
Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, USA
*To whom correspondence should be addressed
Received June 18, 1990. Revised August 16, 1990. Accepted August 16, 1990.
The preparation of fully protected diisopropylamino-ß-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity > 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-/Bu (Gua), ß-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic ‘Hammerhead Ribozyme’. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Takemoto, L. L. Spremulli, L. A. Benkowski, T. Ueda, T. Yokogawa, and K. Watanabe Unconventional decoding of the AUA codon as methionine by mitochondrial tRNAMet with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system Nucleic Acids Res., April 1, 2009; 37(5): 1616 - 1627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Jaikaran, M. D. Smith, R. Mehdizadeh, J. Olive, and R. A. Collins An important role of G638 in the cis-cleavage reaction of the Neurospora VS ribozyme revealed by a novel nucleotide analog incorporation method RNA, May 1, 2008; 14(5): 938 - 949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Rosen, B. S. Brooks, E. Guth, C. S. Francklyn, and K. Musier-Forsyth Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs RNA, July 1, 2006; 12(7): 1315 - 1322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. LEWDOROWICZ, Y. YOFFE, J. ZUBEREK, J. JEMIELITY, J. STEPINSKI, R. KIERZEK, R. STOLARSKI, M. SHAPIRA, and E. DARZYNKIEWICZ Chemical synthesis and binding activity of the trypanosomatid cap-4 structure RNA, September 1, 2004; 10(9): 1469 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Carrasco, Y. Buzin, E. Tyson, E. Halpert, and Z. Huang Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion Nucleic Acids Res., March 8, 2004; 32(5): 1638 - 1646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Iwatani, A. E. Rosen, J. Guo, K. Musier-Forsyth, and J. G. Levin Efficient Initiation of HIV-1 Reverse Transcription in Vitro. REQUIREMENT FOR RNA SEQUENCES DOWNSTREAM OF THE PRIMER BINDING SITE ABROGATED BY NUCLEOCAPSID PROTEIN-DEPENDENT PRIMER-TEMPLATE INTERACTIONS J. Biol. Chem., April 11, 2003; 278(16): 14185 - 14195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Tuite, J. C. Shiels, and A. M. Baranger Substitution of an essential adenine in the U1A-RNA complex with a non-polar isostere Nucleic Acids Res., December 1, 2002; 30(23): 5269 - 5275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Shiels, J. B. Tuite, S. J. Nolan, and A. M. Baranger Investigation of a conserved stacking interaction in target site recognition by the U1A protein Nucleic Acids Res., January 15, 2002; 30(2): 550 - 558. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Feinberg and S. Joseph Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation PNAS, September 13, 2001; (2001) 211184098. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Q. Sun, M. J. Cairns, E. G. Saravolac, A. Baker, and W. L. Gerlach Catalytic Nucleic Acids: From Lab to Applications Pharmacol. Rev., September 1, 2000; 52(3): 325 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Nagan, P. Beuning, K. Musier-Forsyth, and C. J. Cramer Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelixAla variants Nucleic Acids Res., July 1, 2000; 28(13): 2527 - 2534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Schmidt, R. Welz, and S. Muller RNA double cleavage by a hairpin-derived twin ribozyme Nucleic Acids Res., February 15, 2000; 28(4): 886 - 894. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Fischer, P. J. Beuning, and K. Musier-Forsyth Identification of Discriminator Base Atomic Groups That Modulate the Alanine Aminoacylation Reaction J. Biol. Chem., December 24, 1999; 274(52): 37093 - 37096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hwang, N. Tamilarasu, K. Ryan, I. Huq, S. Richter, W. C. Still, and T. M. Rana Inhibition of gene expression in human cells through small molecule-RNA interactions PNAS, November 9, 1999; 96(23): 12997 - 13002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. O. Merlo, N. Cowen, T. Delate, B. Edington, O. Folkerts, N. Hopkins, C. Lemeiux, T. Skokut, K. Smith, A. Woosley, et al. Ribozymes Targeted to Stearoyl–ACP {Delta}9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves PLANT CELL, October 1, 1998; 10(10): 1603 - 1622. [Abstract] [Full Text]
|
|
|
|
|
|
Q. Yu, D. B. Pecchia, S. L. Kingsley, J. E. Heckman, and J. M. Burke Cleavage of Highly Structured Viral RNA Molecules by Combinatorial Libraries of Hairpin Ribozymes. THE MOST EFFECTIVE RIBOZYMES ARE NOT PREDICTED BY SUBSTRATE SELECTION RULES J. Biol. Chem., September 4, 1998; 273(36): 23524 - 23533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Beuning, F. Yang, P. Schimmel, and K. Musier-Forsyth Specific atomic groups and RNA helix geometry in acceptor stem recognition by a tRNA synthetase PNAS, September 16, 1997; 94(19): 10150 - 10154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Hertel, A. Peracchi, O. C. Uhlenbeck, and D. Herschlag Use of intrinsic binding energy for catalysis by an RNA enzyme PNAS, August 5, 1997; 94(16): 8497 - 8502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Beigelman, J. A. McSwiggen, K. G. Draper, C. Gonzalez, K. Jensen, A. M. Karpeisky, A. S. Modak, J. Matulic-Adamic, A. B. DiRenzo, P. Haeberli, et al. Chemical Modification of Hammerhead Ribozymes J. Biol. Chem., October 27, 1995; 270(43): 25702 - 25708. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Triana-Alonso, K. Chakraburtty, and K. H. Nierhaus The Elongation Factor 3 Unique in Higher Fungi and Essential for Protein Biosynthesis Is an E Site Factor J. Biol. Chem., September 1, 1995; 270(35): 20473 - 20478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Triana-Alonso, M. Dabrowski, Jör. Wadzack, and K. H. Nierhaus Self-coded 3`-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase J. Biol. Chem., March 17, 1995; 270(11): 6298 - 6307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Strobel and T. Cech Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site Science, February 3, 1995; 267(5198): 675 - 679. [Abstract] [PDF]
|
|
|
|
 |
|
 S Joseph, A Berzal-Herranz, B M Chowrira, S E Butcher, and J M Burke Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes & Dev., January 1, 1993; 7(1): 130 - 138. [Abstract] [PDF]
|
|
|
|
|
|
A Berzal-Herranz, S Joseph, and J M Burke In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes & Dev., January 1, 1992; 6(1): 129 - 134. [Abstract] [PDF]
|
|
|
|
|
|
K Musier-Forsyth, N Usman, S Scaringe, J Doudna, R Green, and P Schimmel Specificity for aminoacylation of an RNA helix: an unpaired, exocyclic amino group in the minor groove Science, August 16, 1991; 253(5021): 784 - 786. [Abstract] [PDF]
|
|
|
|
|
|
W. Pieken, D. Olsen, F Benseler, H Aurup, and F Eckstein Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes Science, July 19, 1991; 253(5017): 314 - 317. [Abstract] [PDF]
|
|
|
|
|
|
B. Sargueil, J. McKenna, and J. M. Burke Analysis of the Functional Role of a G{middle dot}A Sheared Base Pair by in Vitro Genetics J. Biol. Chem., October 6, 2000; 275(41): 32157 - 32166. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Bernal-Mendez and C. J. Leumann Conformational Diversity Versus Nucleic Acid Triplex Stability, a Combinatorial Study J. Biol. Chem., September 14, 2001; 276(38): 35320 - 35327. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Feinberg and S. Joseph Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation PNAS, September 25, 2001; 98(20): 11120 - 11125. [Abstract] [Full Text] [PDF]
|
|