Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Abstract

Modified oligonucleotides containing sulphur group have been useful tools for
studies of carcinogenesis, protein or nucleic acid structures and functions,
protein-nucleic acid interactions, and for antisense modulation of gene
expression. One successful example has been the synthesis and study of
oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6-
mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia.
During the research of its toxicity, a method was developed to use the sulphur
group as a versatile position for post-synthetic modification.
The advantage of application of post-synthetic modification lies in its
convenience. Synthesis of oligomers with normal sequences has become
routine work in most laboratories. However, design and synthesis of a proper
phosphoramidite monomer for a new modified nucleoside are always difficult
tasks even for a skilful chemist. Thus an alternative method (post-synthetic
method) has been invented to overcome the difficulties. This was achieved by
incorporation of versatile nucleotides into oligomers which contain a leaving
group, that is sufficiently stable to withstand the conditions of synthesis but
can be substituted by nucleophiles after synthesis, to produce, a series of
oligomers each containing a different modified base. In the current project, a
phosphoramidite monomer with 6-thioguanine has been successfully
synthesised and incorporated into RNA. A deprotection procedure, which is
specific for RNA was designed for oligomers containing 6-thioguanosine. The
results were validated by various methods (UV, HPLC, enzymatic digestion).
Pioneer work in utilization of the versatile sulphur group for post-synthetic
modification was also tested.
Post-synthetic modification was also carried out on DNA with 6-
deoxythioguanosine. Electrophilic reagents with various functional groups
(alphatic, aromatic, fluorescent) and bi-functional groups have been attached
with the oligomers.

Date of Award	Jun 2003
Original language	English
Supervisor	Qinguo Zheng (Supervisor) & Eric Lattmann (Supervisor)