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Synthesis of modified ribonucleosides and oligoribonucleotide hybridization probes


Lewis, Michael Robert (1988) Synthesis of modified ribonucleosides and oligoribonucleotide hybridization probes. Master's thesis, California Institute of Technology.


Two strategies for the construction of chemically modified, DNA cleaving oligoribonucleotide probes are described. In the first strategy, the synthetic substrate 5-(3-aminoallyl)uridine 5'-triphosphate (AA-UTP, 1) is reacted with RNA polymerase II to synthesize an aminoallyl-oligoribonucleotide, and the DNA cleaving moiety is introduced by nonspecific, posttranscriptional derivatization of the primary amine groups of the modified RNA. The second strategy involves the incorporation of a modified ribonucleoside 3',5'-bisphosphate, covalently tethered to a DNA cleaving functionality, into an oligoribonucleotide by reaction with T4 RNA ligase. A general method for the chemical synthesis of ribonucleoside 3',5'-bisphosphates was developed. Using this method, uridine 3',5'-bisphosphate (pUp, 2) was synthesized in six steps with an overall yield of 14%. A modified pUp derivative, 5-(3-(3-nitrophenoxy)acetamidoally1)-, uridine 3',5'-bisphosphate (NA-pUp, 12), was also synthesized with an overall yield of 3% for nine steps. The RNA polymerase substrate 1 was prepared by known methods and tested for compatibility with 17 RNA polymerase II in an in vitro "run-off' transcription system. The incorporation of AA-UTP by 17 RNA polymerase was found to be less efficient than that of uridine 5'-triphosphate (UTP), in contrast to results reported for SP6 RNA polymerase. In vitro transcription synthesis of oligoribonucleotides and aminoallyl-oligoribonucleotides by SP6 RNA polymerase gave irreproducible results. In the Ti RNA polymerase system, transcription synthesis produced heterogeneous mixtures of oligoribonucleotides or aminoallyl-oligoribonucleotides. Analysis of the transcription products by high-resolution gel electrophoresis showed that the distribution of transcripts did not vary significantly with buffer conditions or template end structure.

Item Type:Thesis (Master's thesis)
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Unknown, Unknown
Thesis Committee:
  • Unknown, Unknown
Defense Date:29 September 1987
Record Number:CaltechTHESIS:04132010-091809823
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5717
Deposited By: Tony Diaz
Deposited On:17 Jun 2010 16:16
Last Modified:26 Dec 2012 03:24

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