Antisense Oligonucleotides and RNA Interference - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 344

[109] . However, due to the negatives associated with first-generation AS ODNs, such
as large size and chemical and enzymatic instability, second- and third-generation AS
ODNs were developed.
7.6.2 Second-Generation AS ODNs
These include RNA oligonucleotides with alkyl modifications at the 2 position of the
ribose sugar such as 2- O -methyl RNA (OMe-RNA) and 2- O -methoxyethyl RNA
(MOE-RNA) and were synthesized by replacing the 2-OH group with a methyl or a
methoxyethyl group, respectively ( Fig. 7.6B ). These oligonucleotides were designed
to address issues like nonspecific protein binding and cytotoxicity associated with
phosphorothioate AS ODNs, and they are more resistant to nucleases than phospho-
rothioates. However, the major drawbacks associated with these oligonucleotides are
their poor elimination properties and RNase H-independent antisense mechanism of
action [110] . These agents are only effective through the steric blockade mechanism.
7.6.3 Third-Generation AS ODNs
These include gapmer AS ODNs like PNAs, LNAs, N3-P5 phosphoroamidate (PA),
HNAs, 2-F-arabino nucleic acids, cyclohexene nucleic acid, caged nucleic acids,
and others, as shown in Fig. 7.6C . A gapmer contains a central block of deoxynu-
cleotides sufficient to induce RNase H cleavage flanked by blocks of 2- O -methyl-
modified ribonucleotides that protect the internal block from nuclease degrada-
tion [111] . These AS ODNs have increased thermal stability in hybridization and
enhanced target recognition but do not support RNase H activity. These are also com-
paratively less toxic than first- or second-generation oligonucleotides as they show
low interaction with plasma proteins.
One of the earliest and most studied third-generation constructs for antisense
are PNAs . PNAs are AS ODNs in which the sugar phosphate backbone is replaced
completely by polyamide linkages comprising repeating N -(2-aminoethyl)glycine
units attached to nucleobases via methylene carbonyl linkers [111,112] . These pos-
sess increased stability and favorable hybridization [113] due to absence of negative
charges on the PNA oligomers, but do not support the RNase H antisense mechanism.
These exert antisense effect through steric blockade and can bind to both RNA and
transcription factors [114,115] . N3-P5 PA morpholino-oligonucleotides are synthe-
sized by substituting the deoxyribose moiety with a morpholino ring, and the charged
phosphodiester linkage with a neutral PA linkage [116] . These are biologically stable
[117] and possess efficient antisense activity that is RNase H independent. These are
comparatively less toxic than first- or second-generation oligonucleotides and show
low interaction with plasma proteins. LNA is a new and promising third-generation
modification composed of nucleotides that are “locked” into a single conformation
via a 2-O, 4-C methylene linkage in 1,2:5,6-di- O -isopropylene--D-allofuranose
[117] . These possess remarkably increased thermodynamic stability and enhanced
nucleic acid recognition.