Synthesis of modified ribonucleosides and oligoribonucleotide hybridization probes

Citation

Lewis, Michael Robert (1988) Synthesis of modified ribonucleosides and oligoribonucleotide hybridization probes. Master's thesis, California Institute of Technology. doi:10.7907/0x5t-me74. https://resolver.caltech.edu/CaltechTHESIS:04132010-091809823

Abstract

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.

Thesis Files