Studies on modified oligonucleotides

Article abstract of DOI:10.1080/10426500701734539

A series of isonucleotide incorporated antisense oligodeoxynucleotides have been synthesized, in which isonucleotide was introduced at different positions of the sequences. The results showed that though the incorporated isonucleotides at different position of the sequence interfered in the binding ability in different extent, B-form duplexes were maintained and the binding abilities of the 3′-end modified duplexes were better than the corresponding mismatched duplexes. The DNA/RNA hybrid formed by modified oligodeoxynucleotide and its target RNA could activate the activity of RNase H. The 3′-end modified antisense oligodeoxynucelotides showed the inhibition on S glycoprotein expression of SARS-CoV at the mRNA levels in insect Sf9 cells. In this presentation, a novel class of amino-isonucleoside was synthesized and incorporated into different positions in the sense or antisense strand of siRNA duplexes. The 3′ and 5′ sense strand aminonucleoside modified siRNAs (ssISO-siRNA1 and ssISO-siRNA2) showed the similar duplex thermal and serum stability as natural one and the luciferase activities showed that such modified siRNA is compatible with the intracellular siRNA machinery. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500701734539

Phosphorus, Sulfur, and Silicon, 183:326–334, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701734539
Studies on Modified Oligonucleotides
Li He Zhang, Zhen Jun Yang, and Liang Ren Zhang
State Key Laboratory of Natural & Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University, Beijing, China
A series of isonucleotide incorporated antisense oligodeoxynucleotides have been
synthesized, in which isonucleotide was introduced at different positions of the
sequences. The results showed that though the incorporated isonucleotides at dif-
ferent position of the sequence interfered in the binding ability in different extent,
B-form duplexes were maintained and the binding abilities of the 3ꢀ-end modified
duplexes were better than the corresponding mismatched duplexes. The DNA/RNA
hybrid formed by modified oligodeoxynucleotide and its target RNA could acti-
vate the activity of RNase H. The 3ꢀ-end modified antisense oligodeoxynucelotides
showed the inhibition on S glycoprotein expression of SARS-CoV at the mRNA lev-
els in insect Sf9 cells. In this presentation, a novel class of amino-isonucleoside
was synthesized and incorporated into different positions in the sense or anti-
sense strand of siRNA duplexes. The 3ꢀ and 5ꢀ sense strand aminonucleoside
modified siRNAs (ssISO-siRNA1 and ssISO-siRNA2) showed the similar du-
plex thermal and serum stability as natural one and the luciferase activities
showed that such modified siRNA is compatible with the intracellular siRNA
machinery.
Keywords Isonucleoside; aminonucleoside; siRNA; antisense oligodeoxynucelotide
INTRODUCTION
The rapid and specific inhibition of gene product expression makes an-
tisense technology appealing for therapeutic applications and analysis
of the function of newly discovered genes1 and since the term RNA
interference (RNAi) was initially introduced by Fire and coworkers,2
RNAi has been found to be a very powerful tool for gene regulation.
This work was supported by the National Natural Science Foundation of China
(20332010 and SFCBIC 20320130046), the Ministry of Science and Technology of China
(2005BA711A04) and Foundation for the Author of National Excellent Doctoral Disser-
tation of PR China (FANEDD200265).
Address correspondence to Li He Zhang, State Key Laboratory of Natural &
Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xue-Yan
Road, Beijing 100083, China. E-mail: xbna@pku.edu.cn
326

Studies on Modified Oligonucleotides
327
The sequence-selective gene inhibition by siRNA and its rapid adop-
tion as a powerful tool in cell culture has generated the expectation
for its use to improve target therapeutics for cancer, metabolic, inflam-
matory, infectious, neurological and other types of diseases.3 Although
siRNA holds promise for combating human diseases, siRNA therapeu-
tics is hindered by poor intracellular uptake, limited blood stability
and non-specific immune stimulation. Several requirements must be
fulfilled by a potential antisense oligonucleotide or siRNA including
rapid penetration into cells, stability against the degradation by nucle-
ase, delivery methods and dosing changes.4−7 To improve the biosta-
bility of oligonucleotides, chemical modification is widely used. Among
the modifications for antisense oligonucleotides, the phosphorothioate
oligodeoxynucleotide is most frequently used, which is resistant to nu-
clease digestion, is recognized by RNase H, and is easily mass-produced.
Though phosphorothioate oligodeoxynucleotides are currently used as
the standard choice of chemically modified oligodeoxynucleotides, its
non-sequence-specific protein binding leads to significant side effects,
including complement activation, thrombocytopenia, inhibition of cell-
matrix interaction, or reduction of cell proliferation. To overcome these
limitations, intensive efforts were focused on the development of other
kinds of chemically modified oligonucleotides, for example, modifica-
tions of the 2ꢀ-position of riboses with electronegative substituents such
as the 2ꢀ-O-(2-methoxy)ethyl (MOE) group8 or a 2ꢀ-O,4ꢀ-C-methylene
bridge (locked nucleic acid; LNA) were performed.9 But on the other
hand, the RNA-like conformation leads to the disappearance of the
oligonucleotideꢀs ability to activate RNase H, which is considered to
be an important pathway of antisense action. In siRNA applications,
modification of the base moiety is relatively less attempted. Most of
the chemical modifications of oligonucleotide were done on the 2ꢀ-OH
group of the ribose moiety. It was found that modifications at the 2ꢀ-
position of pentose sugars in siRNAs are in general well tolerated.10
Since such ribose modifications confer an RNA-like conformation to the
oligonucleotide, they increase the target RNA binding affinity and cel-
lular uptake.
STUDIES ON THE SYNTHESIS AND HYBRID PROPERTIES
OF ANTISENSE OLIGONUCLEOTIDE CONTAINING
ISONUCLEOSIDES
Isonucleosides represent a novel class of nucleoside analogues in which
the nucleobase is linked to various positions of ribose other than C-1.
Because of the shift of N-glycosidic bond, the chemical and enzymatic

328
L. H. Zhang et al.
FIGURE 1 The structures of 1ꢀ,4ꢀ-anhydro-2ꢀ–deoxy-2ꢀ-nucleobase-L-
arabinitol, 1ꢀ,4ꢀ-anhydro-2ꢀ-deoxy-2ꢀ–nucleobase -D-arabinitol, and 4-deoxy-4-
nucleobase-2,5-anhydro-L- mannitols (A) 4-deoxy-4-nucleobase-2,5-anhydro-D-
mannitols (B).
stabilities of the nucleoside are increased. Nair reported the synthe-
sis of isonucleoside, 1ꢀ,4ꢀ-anhydro-2ꢀ-deoxy- 2ꢀ-nucleobase-D-arabinitol
(Figure 1), and found that oligodeoxynucleotides containing such isonu-
cleoside could form a duplex with complementary DNA sequence and
antagonize the degradation of enzyme.11
We reported the synthesis of 1ꢀ,4ꢀ-anhydro-2ꢀ-deoxy-2ꢀ-nucleobase-
L-arabinitol (Figure 1), the stereoisomer of 1ꢀ,4ꢀ-anhydro-2ꢀ-deoxy-
2ꢀ-nucleobase-D-arabinitol. However, the oligonucleotide containing
1ꢀ,4ꢀ-anhydro-2ꢀ-deoxy-2ꢀ-nucleobase-L-arabinitol could not form regu-
lar base pair as described for its D-form partner. Since an RNA-like
conformation of the modified oligonucleotide could increase the target
RNA binding affinity and cellular uptake, we designed and synthe-
sized 4-deoxy-4-nucleobase-2,5-anhydro-D-mannitols (B) and 4-deoxy-
4-nucleobase-2,5-anhydro-L-mannitols (A) which contain an additional
hydroxymethyl group at the sugar moiety.(Figure 1). The hybrid prop-
erties of the oligodeoxynucleotides incorporated with A or B with the
complementary DNA were investigated.
The different linkage of the phosphate backbone in the oligonu-
cleotide incorporated with isonucleoside B could influence the hybrid
property with the complementary strand (Figure 2). Oligomer II (with
6ꢀ → 4ꢀ linkage ) failed to hybridize with d(A)14, nevertheless, a stable
FIGURE 2 The different linkage of the phosphate backbone in the oligonu-
cleotide incorporated with isonucleoside B.

Studies on Modified Oligonucleotides
329
duplex of oligomer I (with 1ꢀ → 4ꢀ linkage) with d(A)14 was formed. Com-
puter simulation showed that in I/d(A)14 the Cꢀ6 hydroxyl group of each
unit is located in the groove area when hybridized to the complementary
strand where it can form hydrogen bonds with water in the medium,
while in II/ d(A)14 most of Cꢀ1 hydroxyl groups are directed to the in-
side of the duplex.12 In the case of the oligonucleotide incorporated with
isonucleoside A, the existence of hydroxymethyl group on the isonucleo-
side could stabilize the formed duplex with its complementary sequence,
moreover, the octaoligoisonucleotide (isoT)2(isoG)4(isoT)2 consisting of
4-deoxy-4-nucleobase-2,5-anhydro-L-mannitols (A) could form a paral-
lel intermolecular G-quadruplex structure, K+, Na+ and Li+ (K+ >Na+
> Li+) can prompt the formation of G-quartet structures and stabilize
them.13
The building block containing 4-deoxy-4-nucleobase-2,5-anhydro-L-
mannitols (A) was synthesized and then formed the oligoisonucleotides
I–IV as shown in Figure 3.
The hybrid properties of oligomers I–IV showed that when the
isonucleotide was located at 3ꢀ-end, the duplex was more stable
than the corresponding mismatched sequences, whereas when isonu-
cleotide was located at the center of the sequence, the duplex was
less stable than the corresponding single-nucleoside-mismatched se-
quence but was more stable than multinucleoside-mismatched se-
quence (Table I). Circular dichroism (CD) spectra showed that the
hybrid duplex formed by modified oligodeoxynucleotides and their
complementary sequence possessed very similar conformations, the
single isonucleotide modified oligodeoxynucleotides formed typical
TABLE I Tm Value of the Duplexes of Isonucleoside
Modified Oligonucleotides with the
Complementary DNA Sequence
Duplexes
Tm (◦C)
ꢀTm(◦C)
I/VII
II/VI
59.3
54.2
58.6
54.1
61.5
58.0
58.0
49.1
56.2
44.3
37.1
−2.2
−7.7
−2.9
−7.4
—
−3.5
−3.5
−12.4
−5.3
−17.2
−24.4
III/VI
IV/VI
V/VI
VII/VI
VIII/VI
IX/VI
X/VI
XI/VI
XII/VI

330
L. H. Zhang et al.
FIGURE 3 The sequences of isonucleotide-incorporated oligodeoxynu-
cleotides.
B-form duplexes such as natural oligodeoxynucleotides. The com-
puter simulation results also showed that B-form duplexes were
maintained during the entire course of simulation, the helical pa-
rameters for the modified oligonucleotide duplexes were very simi-
lar to the native DNA duplex. The averaged structure of the native
DNA duplex was overlapped with the isonucleotide-modified duplexes,
respectively.
The above result indicated that the incorporation of isonucleotide
into the oligodeoxynucleotide might only lead to the local conforma-
tional change of the duplex. The DNA/RNA hybrid formed by a mod-
ified oligoisonucleotide and its target RNA could activate RNase H.
The 3ꢀ-end modified antisense oligoisonucelotides I and III inhibited

Studies on Modified Oligonucleotides
331
S-glycoprotein expression of SARS-CoV at the mRNA levels in insect
Sf9 cells.14
STUDIES ON THE SYNTHESIS, HYBRID AND SILENCING
PROPERTIES OF SIRNA CONTAINING
AMINO-ISONUCLEOSIDES IN SENSE OR
ANTISENSE STRAND
For aminoisonucleotide-modified oligonucleotides, introduction of
amino groups may increase their thermal stability via the strong bind-
ing of cationic amino groups with negative charged phosphate back-
bones of the complementary sequences. Building blocks 7 and 13 for
solid phase oligonucleotide synthesis were synthesized from starting
materials 1 and 2 (22), respectively (Scheme 1). We developed a one-step
method that at room temperature compound 1 or 2 was directly reacted
with sodium azide in the presence of triphenyl phosphine and carbon
tetrabromide, giving the key intermediate 3 or 8 in good yield.15 Incu-
bation of compound 3 or 8 with 1% HCl followed by sodium hydroxide
and sodium borohydride in second one-pot reaction at 0◦C gave unpro-
tected azido isonucleoside 4 or 9 at yield of 84% and 76%, respectively.
After the sequential protection and deprotection, the obtained building
blocks 7 and 13 were finally incorporated into the sequences of oligonu-
cleotides according to the solid phase protocols. Amino-isonucleoside
building blocks 7 and 13 were incorporated into the sense or antisense
strand of a designed siRNA.
SCHEME 1 Synthesis of amino-isonucleoside building block.

332
L. H. Zhang et al.
TABLE II Sequences and Tm Values of Native siRNA
(siRNA-NC) and Amino-Isonucleoside Modified Sense
Strand ( ssISO-siRNA 1-5)
Name
Sequence
Tm(◦C)
siRNA-NC
5ꢀ-CUGGUAAAGCAUUCAGUAUtt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5ꢀ
5ꢀ-CUGGUAAAGCAUUCAGUATtt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5ꢀ
5ꢀ-CTGGUAAAGCAUUCAGUAUtt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5ꢀ
5ꢀ-CUGGUAAAGCAUUCAGUAUtt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5
5ꢀ-CTGGUAAAGCAUUCAGUAUTt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5ꢀ
5ꢀ-CTGGUAAAGCAUUCAGUATt-3ꢀ
3ꢀ-ttGACCAUUUCGUAAGUCAUA-5ꢀ
66
ssISO-siRNA(1)
ssISO-siRNA(2)
ssISO-siRNA(3)
ssISO-siRNA(4)
ssISO-siRNA(5)
66
66
62
56
36
It was found that incorporation of one amino-isonucleoside at 3ꢀ or
5ꢀ terminal of modified oligonucleotides exhibited no effect on the du-
plex thermal stability which is equal to native siRNA form, siRNA NC.
However, internal incorporation of one amino-isonucleoside into siRNA
duplexes decreased the thermal stability. Furthermore, when two or
more amino-isonucleosides were incorporated into siRNAs, Tm values
decreased strikingly (Table II).
Incubation of modified siRNAs with 50% FBS at 37◦C indicated
that siRNAs with a single amino-isonucleoside at the sense strand,
such as ssISO-siRNA1, ssISO-siRNA2, and ssISO-siRNA3, exhibited
the similar serum stability as their native siRNA with all degraded
in 3 h. Modified siRNAs with two or more amino-isonucleosides at
the sense strand, such as ssISO-siRNA4 and ssISO-siRNA5, showed
further weaker serum stability compared with the native siRNA and
rapidly degraded within 1.5 h. The firefly luciferse-targeting modified
siRNAs with amino-isonucleosides in either the sense strand or an-
tisense strand were evaluated based on a dual-luciferase assay that
renillar luciferase acts as an internal control. We found that sense
strand-modified siRNAs with a single amino-isonucleoside at the 3ꢀ-
or 5ꢀ-end, such as ssISO-siRNA1 and ssISO-siRNA2, exhibited com-
parative and even better activity than the native siRNA NC (Table III).
Alternative modifications with amino-isonucleoside at the siRNA inter-
nal region, such as ssISO-siRNA3, bring some negative effect on siRNA
activity compared with the terminal modification, and such negative ef-
fect become further apparent when internal and terminal modification
in the sense strand were combined together (Table III). In contrast to

Studies on Modified Oligonucleotides
333
TABLE III Firefly Renilla Luciferase Activities of siRNA, Different
ssISO-siRNA and asISO-siRNA
All experiments were performed in triplicates and repeated at least twice.
sense strand modifications, antisense strand modification led to more
negative effect on the silencing activity of siRNA duplex. A single nu-
cleotide modification at the 3ꢀ or 5ꢀ-end of the antisense strand reduced
the siRNA silencing activity to 40%–50%. Internal modification of the
antisense strand of siRNA has more negative effect on the silencing
activity than the terminal modification. Terminal modification at both
the 3ꢀ- and 5ꢀ-end of the antisense strand reduced the silencing activity
of the siRNA significantly but does not abolish the silencing activity
completely. However, combination of the terminal and internal modifi-
cations on antisense strand totally abolish the silencing activity.
Here, we conclude that siRNA sense strand modification has less ef-
fect on siRNA activity while antisense strand modification significantly
reduces siRNA activity. Our data also suggests: 1) the bases in modi-
fied siRNAs, despite of their lower activity, retain their hybridization
properties with complementary sequences; 2) the torsion angles in the
sugar-phosphate backbones would not perturb the entry of the modified
siRNA into RISC; and 3) the two strands of the siRNA duplex are not
equally eligible for assembly into RISC.
Isonucleoside modified oligonucleotide is superior to other chemical
modifications since such modified RNAs are good substrates of RNase
H and isonucleoside triphosphates can be recognized by many differ-
ent DNA polymerases.16 Furthermore, siRNA sense strand modifica-
tion with amino-isonucleoside at the 3ꢀ or 5ꢀ-end showed comparable

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L. H. Zhang et al.
gene-silencing activity and serum stability as their native siRNA.
Taken together, isonucleoside modified oligonucleotide represents a
novel direction and is a good mimic of natural oligonucleotide with the
required biological and chemical properties.
REFERENCES
[1] E. Uhlman and A. Peyman, Chem. Rev., 90, 543 (1990).
[2] A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello, Nature,
391, 806 (1998).
[3] D. Zhou, Q. S. He, C. Wang, J. Zhang, and F Wong-Staal, Current Topics in Medicinal
Chemistry, 6, 901 (2006).
[4] R. W. Wagner, Nature, 372, 333 (1994).
[5] S. T. Crooke, Curr. Opin. Biotechnol., 3, 656 (1992).
[6] V. V. Vlasov, L. A. Balakireva, and L. A. Yakubov, Biochim. Biophys. Acta, 1197, 95
(1997).
[7] A. M. Krieg, J. Tonkinson, S. Matson, Q. Zhao, M. Saxon, L. Zhang, U. Bhanja, L.
Yakubov, and C. A. Stein, Proc. Natl. Acad. Sci. USA, 90, 1048 (1993).
[8] B. F. Baker, S. S. Lot, T. P. Condon, S. Cheng-Flournoy, E. A. Lesnik, H. M. Sasmor,
and C.F. Bennett, J. Biol. Chem., 272, 11994 (1997).
[9] C. Wahlestedt, P. Salmi, L. Good, J. Kela, T. Johnsson, T. Hokfelt, C. Broberger, F.
Porreca, J. Lai, K. Ren, M. Ossipov, A. Koshkin, N. Jakobsen, J. Skouv, H. Oerum,
M. H. Jacobsen, and J. Wengel, Proc. Natl. Acad. Sci. U.S.A., 97, 5633 (2000).
[10] C. R. Allerson, N. Sioufi, R. Jarres, T. P. Prakash, N. Naik, A. Berdeja, L. Wanders,
R. H. Griffey, E. E. Swayze, and B. Bhat, J. Med. Chem., 48, 901 (2005).
[11] T. Wenzel, V. Nair, Bioorg. Med. Chem. Lett., 7, 3195 ( 1997); (b) T. Wenzel and
V. Nair, Bioconjugate Chem.,9, 683 (1998).
[12] L. Zhen, L. Zhang, L. R. Zhang, J. Chen, J. M. Min, and L. H. Zhang, Nucleic Acids
Research, 29, 1470 (2001).
[13] J. Cheng, L. R. Zhang, J. M. Min, and L. H. Zhang, Nucleic Acids Research, 30, 3005
(2002).
[14] Z. L. Wang, J. F. Shi, H. W. Jin, L. R. Zhang, J. F. Lu, and L. H. Zhang, Bioconjugate
Chemistry,16, 1081 (2005).
[15] Z. S. Li, R. P. Qiao, Z. J. Yang, L. R Zhang, and L. H. Zhang, Tetrahedron:Asymmetry,
17, 1056 (2006).
[16] C. W. Jiang, B. C. Li, Z. Guan, Z. J. Yang, L. R. Zhang, and L. H. Zhang, Bioorg. &
Med. Chem., 15, 3019 (2007).