Chemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting
Biochemistry, Genetics and Molecular Biology » "Mutagenesis", book edited by Rajnikant Mishra, ISBN 978-953-51-0707-1, Published: August 17, 2012 under CC BY 3.0 license.
The artificial regulation of gene expression is very important for basic studies analyzing unknown biological functions of target genes. The comparison of phenotypes with and without knockdown of the expression levels of target ed genes may allow to reveal unknown biological functions of the target genes. Artificial regulation of gene expression is also important for therapeutic applications to reduce expression level of mutated target genes.
Knockdown of expression level of mutated target genes may also be useful to avoid undesirable effects produced by the mutated target genes.
Antisense and antigene technologies are powerful tools to artificially regulate target gene expression. In antisense technology, a single-stranded oligonucleotide added from outside may bind with target mRNA to form oligonucleotide-RNA duplex. The formed duplex may inhibit ribosome-mediated translation of target mRNA due to its steric hindrance, or RNaseH may cleave target mRNA in the formed duplex, which may result in reduction of expression level of target mRNA in both cases. In antigene technology, a single-stranded homopyrimidine triplex-forming oligonucleotide (TFO) added from outside may bind with homopurine-homopyrimidine stretch in target duplex DNA by Hoogsteen hydrogen bonding to form pyrimidine motif triplex, where T•A:T and C+•G:C base triplets are formed. The formed triplex inhibits RNA polymerase and transcription factors-mediated transcription of target gene due to its steric hindrance, which may result in downregulation of expression level of target gene.
Serious difficulties, such as poor binding ability of added oligonucleotides with target mRNA or target duplex DNA, and low stability of added oligonucleotides against nuclease degradation, may limit practical applications of the antisense and antigene technologies in vivo. Many different chemically modified oligonucleotides have been developed to overcome these serious difficulties. Torigoe and Imanishi developed a novel class of chemical modification of nucleic acids, 2’-O,4’-C-aminomethylene bridged nucleic acid (2’,4’-BNANC), in which 2’-O and 4’-C of the sugar moiety are bridged with the aminomethylene chain (see paper). 2’,4’-BNANC-modified oligonucleotides showed higher binding affinity with target mRNA, stronger binding ability with target duplex DNA to form triplex, and higher stability against nuclease degradation than the corresponding unmodified oligonucleotides. These excellent properties of 2’,4’-BNANC-modification of oligonucleotides may be favorable for their practical applications to the antisense and antigene technologies.