Design, Synthesis & Evaluation of Potential Antisense Oligonucleotides
(AONs)
Recent times, much attention has been focused to develop antisense
oligonucleotides (AONs) as therapeutic agents. Antisense technology is based on the fact
that, once AONs are targeted to mRNA, they can down regulate the gene expression by
multiple mode of mechanism, which includes the efficient binding of the AON to the
target and/or by the degradation of the target in the heteroduplex by the enzyme RNase H
Towards this direction various modifications of sugar, base and phosphate backbone of
AONs have been attempted (see the Reviews below). But in most of the cases the
modified AONs fails to activate RNase H, which is considered to be an important
pathway of antisense action.
Phosphorothioates, which had emerged as the first generation of antisense drugs,
fails to gather momentum because of its non-antisense effects: high affinity to heparinbinding proteins and low sequence specificity (see: Stein’s paper).
It has been emerged that to act as a successful antisense drug, AONs should full
fill the following criteria: (i) specific target recognition by Watson-Crick base pairing, (ii)
good structural mimicry to the natural DNA-RNA, (iii) activation of RNase H to promote
the target mRNA cleavage (iv) enhanced cellular uptake, and (iv) enhanced resistance to
various nucleases.
Our interest in this area has prompted us to design, synthesize and evaluate AONs
which recruit RNase H. In this regard our interest is two-fold:
(i) We have recently shown that conjugation of various chromophores at the 5’end [Nucleosides & Nucleotides 18, 2785-2818 (1999)] of AON can enhance the binding
affinity to target RNA without altering the global helical conformation or flexibility of
the AON/RNA duplex, keeping the RNase H eliciting power and nuclease resistance in
tact. We .are exploring these conjugated AONs as potential antisense candidates (see
below). (ii) We are also actively involved in the utility of Locked Nucleic Acids, as
RNase H compatible antisense drugs (see below).
Reviews on the Antisense technology:
1) Uhlmann, E.; Peyman, A: Antisense oligonucleotides: A new therapeutic principle.
Chemical Reviews 1990, 90: 543-584.
2) Mesmaekar, A.D.; Haner, R.; Martin, P.; Moser, E.H.. Antisense oligonucleotides.Acc. Chem. Res. 1995, 28:366-374
3) Frier, S.M.; Altmann, K-H. .The ups and downs of nucleic acid duplex
stability:structure stability studies on chemically-modified DNA:RNA duplexes.
Nucleic Acids Res. 1997, 25:4429-4443
4) Crooke, S. T. Progress in Antisense Technology: The End of the Beginning.2000,
Methods in Enzymology, 313, 3-45.
5) Zamaratski, E., Pradeepkumar, P. I., Chattopadhyaya, J. A critical curvey of the
structure- function of antisense oligonucleotide/RNA hetroduplex as a substrate for
RNase H. 2001, .J Biochem Biophys Methods., 48, 189-208. (PDF 315).
6) Stein, C. A.; The experimental use of antisense oligonucleotides: a guide for the
perplexed. J. Clin. Invest. 2001, 108, 641-644.
(1) Tethering of Various Chromophores/ hydrophobic moieties at 3’ and or 5’ end of
AONs.
We have tethered various chromophores having different geometry, bulk, electron
density, polarisability as well as hydrophobicity like phenazine, phenazinium, anthracene,
pyrene (PDF 268, PDF 285), dipyridophenazine-dppz- (PDF 305), [Ru (phenanthroline)2
dppz)2+] (PDF 318) to the 3’and/or 5’ end of AONs and their antisense potential has been
evaluated. All of them showed enhanced binding affinity to the target, augmented
stability towards nucleases and recruitment of RNase H in a faster or comparable rate to
the non-conjugated counterpart (PDF 313).
Groups having different hydrophobicities like cholestrol, cholic acid and cholic
acid triacetate have also been conjugated to the 3’-end of the AONs, keeping in mind
their applicability for easy delivery into cells. A detailed kinetic analysis of RNase H
hydrolysis has been performed where various 3’-conjugated AONs were compared with
the non-conjugated native counter part (PDF 330). It has thus emerged that 3’-dppz
tethered AON has the highest value of Vmax/Km ratio, thereby showing its high capability
to recruit the RNase H.
(2) Design and Synthesis of Various Locked Nucleic Acids.
Locked Nucleic Acids having natural phosphate backbones have attracted much
attention recently (See the recent Reviews below), because of its unprecidential binding
affinity to the target. Among the various sugar modified locked nucleic acids there is only
one qualitative report [PNAS, 97, 5633-38 (200)]. regarding the activation of RNase H,
where the target is cleaved relatively at a reduced rate compared to the unmodified
counterpart.
We have however recently shown that introduction of the 1’,2’-locked oxetane
(1',3'-O-anhydro-β-D-psicofurnosyl)thymine) modified nucleoside T (where sugar is
fixed in the North-East conformation) into AONs (PDF 312, 314) can recruit RNase H as
good as the native, and also can give at least 3 fold increment in endonuclease resistance
(PDF 328). We have also analyzed the kinetics of RNase H hydrolysis of these modified
duplexes in detail(PDF 331).
Reviews on Locked Nucleic Acids:
1) Kvaerno, L., Wengel, J. Antisense molecules and furanose conformations-is it really
that simple? 2001, Chem. Commun., 1419-1424.
2) Herdewijn, P. Targeting RNA with conformationally restricted oligonucleotides
Leibiggs Ann.1996, 1337-1348.
3) Herdewijn, P. Conformationally restricted carbohydrate-modified nucleic acids and
antisense technology. Biochimica et Biophysica Acta. 1999, 1489:167-179.