Biomedical Engineering Reference
In-Depth Information
DNA
pre-mRNA
mRNA
Protein
(A)
(B)
(C)
(D)
RNase H
(E)
Figure 7.3
Various strategies available for antisense knockdown. (A) The normal process of
protein synthesis involving transcription and translation. (B) Transcriptional arrest by DNA-
targeted agents. (C) Prevention of mature mRNA formation by pre-mRNA targeting.
(D) Translational arrest by interruption of the translation apparatus. (E) Prevention of
translation by RNase H enzyme.
near the translational initiation codon and the 3 and 5 untranslated regions. RNase
H is found in both the nucleus and the cytoplasm of all cells
[74]
. Its regular func-
tion is to remove RNA primers from Okazaki fragments during DNA replication.
Hence, oligonucleotides that act via RNase H activation must be designed care-
fully. Although the requirement for an AS ODN to inhibit a protein expression is not
known precisely, modifications in sugar moiety such as sugar type and its orienta-
tion are thought to influence RNase H activation
[75-78]
. Modifications that result
in DNA-like oligonucleotides support RNase H activity, while changes resulting in
RNA-like oligonucleotides do not support RNase H activity. Modifications in oli-
gonucleotide backbone also alter RNase H activity
[79-81]
. However, the favored
design is to use chimeric molecules, such as a single ribonucleotide, which can be
bound to its complementary oligonucleotide backbone, which then serves as a sub-
strate for RNase H
[36]
. These RNase H-dependent oligonucleotides can inhibit pro-
tein expression by binding to any region of the complementary mRNA.
7.5.2 Inhibition of 5
Capping
5 capping is an essential step in the protein synthesis cascade, stabilizing and trans-
lating an mRNA into a mature polypeptide sequence
[82]
. Although this is an effec-
tive mechanism of inhibiting mRNA translation, it is not an established mechanism
of action of AS ODNs due to the inaccessibility of the 5 end of mRNA to capping.
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