Investigations on the influence of 2'-O-alkyl modifications on the base pairing properties of oligonucleotides

Article abstract of DOI:10.1016/S0031-6865(97)00041-1

The antisense strategy has gained wide acceptance as a promising drug development concept. Antisense drugs hybridize with selected complementary sequences in a highly specific manner. However, as a main prerequisite for extensive therapeutic use of this new class of drugs, selected structural modifications are required to adjust pharmacokinetic and pharmacodynamic behavior. In continuation of our earlier investigations on 2'-O-modified oligonucleotides (Gotten, M., Oberhauser, B., Brunar, H., Holzner, A., Issakides, G., Noe, C.R., Schaffer, G., Wagner, E., Bimstiel, M.L., 1991. 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. Nucleic Acids Res. 19, 2629-2635; Wagner, E., Oberhauser, B., Holzner, A., Brunar, H., Issakides, G., Schaffner, G., Gotten, M., Knollmuller, M., Noe, C.R., 1991. A simple procedure for the preparation of protected 2'-O-methyl or 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites. Nucleic Acids Res. 19, 5965-5971) further series of modified oligonucleotides containing different modified 2'-O-adenosines have been synthesized. On the one hand linear alkyl moieties of increasing length, on the other hand oxyethylene moieties of corresponding length were introduced at the 2'-O-position of adenosine. Following another approach a cationic charge was introduced by insertion of an aminohexylmodification at the 2'-O-position of adenosine (Brunar, H., Haberhauer, G., Werner, D., Noe, C.R., 1994. 2'-O-Modified oligonucleotides: Synthesis and biophysical analysis. Eur. J. Pharm. Sci. 2, 150). Molecule dynamics simulations had shown that this cationic modification upon duplex formation leads to both intra- and interstrand interactions. To determine the influence of the different modifications, such as cationic charge, alkyl chainlength and introduction of oxygen into the chain, on duplex stability melting temperatures were measured by recording circular dichroism versus temperature.

Full text of DOI:10.1016/S0031-6865(97)00041-1

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.
Pharmaceutica Acta Helvetiae 73 1998 3–10
Investigations on the influence of 2X-O-alkyl modifications on the base
pairing properties of oligonucleotides
)
Doris Werner, Helmut Brunar, Christian R. Noe
Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe-UniÕersitat, Biocenter, Marie-Curie-Straße 9, D-60439 Frankfurt, Germany
¨
Received 1 October 1997; accepted 8 October 1997
Abstract
The antisense strategy has gained wide acceptance as a promising drug development concept. Antisense drugs hybridize with selected
complementary sequences in a highly specific manner. However, as a main prerequisite for extensive therapeutic use of this new class of
drugs, selected structural modifications are required to adjust pharmacokinetic and pharmacodynamic behavior. In continuation of our
X
Ž
earlier investigations on 2 -O-modified oligonucleotides Cotten, M., Oberhauser, B., Brunar, H., Holzner, A., Issakides, G., Noe, C.R.,
Schaffer, G., Wagner, E., Birnstiel, M.L., 1991. 2X-O-methyl, 2X-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonu-
cleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. Nucleic Acids Res. 19, 2629–2635; Wagner, E.,
Oberhauser, B., Holzner, A., Brunar, H., Issakides, G., Schaffner, G., Cotten, M., Knollmuller, M., Noe, C.R., 1991. A simple procedure
¨
X
X
X
.
for the preparation of protected 2 -O-methyl or 2 -O-ethyl ribonucleoside-3 -O-phosphoramidites. Nucleic Acids Res. 19, 5965–5971
further series of modified oligonucleotides containing different modified 2X-O-adenosines have been synthesized. On the one hand linear
alkyl moieties of increasing length, on the other hand oxyethylene moieties of corresponding length were introduced at the 2X-O-position
of adenosine. Following another approach a cationic charge was introduced by insertion of an aminohexylmodification at the
X
X
Ž
2 -O-position of adenosine Brunar, H., Haberhauer, G., Werner, D., Noe, C.R., 1994. 2 -O-Modified oligonucleotides: Synthesis and
.
biophysical analysis. Eur. J. Pharm. Sci. 2, 150 . Molecule dynamics simulations had shown that this cationic modification upon duplex
formation leads to both intra- and interstrand interactions. To determine the influence of the different modifications, such as cationic
charge, alkyl chainlength and introduction of oxygen into the chain, on duplex stability melting temperatures were measured by recording
circular dichroism versus temperature. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: 2X-O-alkyl modifications; Pairing properties; Oligonucleotides
1. Introduction
type of therapy. However, the application of these com-
pounds is not only restricted to spectacular therapies of
cancer or viral diseases, but might even lead to rather
general alternatives in pharmacotherapy. Nowadays many
new drugs result from a systematic search for antagonists
for different receptors. Taking into account that also recep-
tor proteins are encoded by genes, a certainly significant
advance in receptor oriented pharmacotherapy might result
from the development of antisense drugs which suppress
receptor gene expression. Last but not least, short oligo-
nucleotides interact not only with nucleic acids but also
with nucleic acid binding proteins and other proteins, thus
opening the new therapeutic approach of aptamers.
As a result of the great progress in biological and
biochemical research direct action upon genes to correct
malfunctions or defects has become feasible in principle
within the concept of gene therapy. Due to their sequence
specific interaction with target sequences, even rather short
antisense oligonucleotides may be used as drugs in this
Abbreviations: CD: circular dichroism; DNA: deoxyribonucleic acid;
ODN: oligodeoxynucleotide; RP-HPLC: reversed phase HPLC
)
Corresponding author. Tel.: q49-69-79829302; fax: q49-69-
79829352.
0031-6865r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
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.
PII S0031-6865 97 00041-1

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D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
Fig. 1. Selected 2X-O-modifications.
The physicochemical properties of oligonucleotides,
comprises attachment of a variety of different moieties to
the nucleobases or the hydroxy groups of the ribose.
Among these, the modification of oligonucleotides at the
2X-position of the sugar has been shown to enhance nucle-
ase stability.
which differ greatly from the nature of traditionally ap-
plied drugs, have hitherto presented the greatest obstacle in
their application as drugs. They are oligomeric compounds
with a high molecular weight. Due to their polyanionic
character, oligonucleotides do not penetrate lipophilic cell
membranes easily. There is no previous experience on
their pharmacokinetic behavior. The mechanisms of the
distribution and the cellular uptake of ODNs are poorly
understood. Data available indicate that the cellular uptake
is a saturable receptor-mediated endocytosis and depends
on temperature, concentration, length, specific sequences
and modification 1. In addition, the phosphodiester back-
bone is rapidly degraded by serum and cellular nucleases.
Taking all these factors into account, it is not surprising
that in the meantime various approaches have been re-
ported to adjust the properties of ODNs to the therapeuti-
cal requirements.
In continuation of our earlier investigations on 2X-O-
Ž
modified oligonucleotides Cotten et al., 1991; Wagner et
.
al., 1991 we were interested in conducting a systematic
study on the effect of different modifications at the 2X-posi-
tion. A decrease in duplex stability of long chain alkyl
Ž
modifications has been reported by others Sproat et al.,
.
1991 . Therefore the influence of a systematic increase in
alkyl chainlength was studied first. Bearing in mind that
long alkyl chains tend to form coils, which might be
responsible for the loss of pairing properties due to steric
hindrance, we decided to study bioisosteric oxyethylene
Ž
.
chains with corresponding chainlengths Fig. 1a–h . It is
Ž
.
known that due to stereoelectronic effects gauche-effect
such chains tend to adopt rather linear helical conforma-
tions.
Most of the reported ‘first generation’ modifications of
ODNs were changes of the phosphodiester backbone for
isosteric groups. In addition, replacement of the natural
sugars ribose and deoxyribose, respectively by other sugars
Following a reaction sequence worked out earlier by
X
Ž
.
our group Birnstiel et al., 1991 , the 2 -O-modifications
were first attached to adenosine, followed by automated
oligomerization using standard 2-cyanoethyl phospho-
ramidite chemistry with extended coupling times. A series
of modified dodecamers were synthesized, incorporating
up to six 2X-O-alkyl nucleotides within the sequence. Dou-
ble strand geometry and hybridization thermodynamics of
Ž
or rings has been described for a review see Englisch and
.
Gauss, 1991; Reddy, 1996 . A third group of modifications
1 Ž
Erickson and Izant, 1992; Crooke, 1995
.

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D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
5
Fig. 2. Synthesis of pentaethylene glycol monomethylether.
these model compounds were studied systematically. The
effects were compared with those of a zwitterionic modifi-
synthesized and hybridization properties had been investi-
gated.
Ž
.
cation Fig. 1i , which seemed to support double strand
formation.
2. Results and discussion
Such modifications are not unknown in principle. Some
2X-O-alkylmodifications have been attached to adenosine
Ž
2.1. Synthesis of modified building blocks
According to the method developed earlier for the
.
Lesnik et al., 1993 , ODNs had been synthesized and their
double strand geometry and hybridization thermodynamics
were studied. At the time of the present study oxyethylene
moieties were introduced into the 2X-position of thymidine
preparation of 2X-O-methyl and 2X-O-ethyl nucleotides
X
Ž
.
Wagner et al., 1991 , all 2 -O-alkyladenosines were pre-
Ž
pared via direct alkylation of unprotected adenosine Fig.
.
Ž
.
Martin, 1995 . ODNs containing some substituted
Ž
.
thymidines in the middle of the sequences have been
1 . To prepare the modifications a to d Fig. 1 , we used
Table 1
Synthesis of the 2X-O-alkyladenosines reported in Fig. 3
1
X
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Compound
Formula mw
Mp 8C
Yield %
H-NMR: d ppm ; J Hz ; H-C 1
Elemental analysis C, H, N or MS ESI
a
Ž .
5.99 d, Js6
1c
2c
3c
4c
5c
6c
7c
8c
C14 H21N5O4
C17 H27 N5O4
C20 H33N5O4
C26 H45N5O4
C13 H19 N5O5
C15 H23N5O6
156–158
127
117
116
124
oil
48
12
37
18
16.8
22
24
52.28, 6.72, 21.45
b
Ž
.
Ž
.
5.84 d, Js2.6
54.58, 7.4, 18.73 0.5 H2O
a
Ž
Ž
.
5.96 d, Js6
58.98, 8.22, 17.11
66.78, 8.33, 11.83
a
c
.
.
5.95 d, Js4.2
Ž
Ž
.
5.89 d, Js5.9
48.21, 6.18, 18.94 0.3 H2O
a
Ž
.
w
q x
6.0 d, Js4.5
367.9 M
d
Ž .
C17 H27 N5O7
C21H35N5O9
oil
oil
6.0 d, Js4
49.48, 6.5, 16.89
49.73, 7.14, 13.49 0.4 H2O
d
Ž
.
Ž
.
10.7
5.88 d, Js7.68
Solvents: a DMSO-d6, bCDCl3:CD3OD 1:2, cCDCl3:DMSO-d6 2:1, dCDCl3.
Table 2
X
Ž
.
Phosphitylation of protected 2 -O-alkyladenosines Fig. 4
31
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Compound
Formula mw
Mp 8C
Yield %
PyNMR: d ppm ; CDCl3
Elemental analysis C, H, N or MS ESIq
w
xq
6a
6b
6c
6d
6e
6f
C51H60 N7O8 P
C54 H67 N7O8 P
C57 H70 N7O8 P
C63 H84 N7O8 P
45
58
54
44
34
37
45
40
99
85
88
92
92
85
99
92
151.35, 150.65
151.26, 150.58
151.49, 150.77
151.51, 150.76
152.44, 150.92
151.12, 150.64
151.35, 150.8
151.09, 150.64
930.06 MqH
w
xq
973.7 MqH
66.64, 6.85, 9.55 0.8 H2O
Ž
Ž
.
.
67.95, 7.67, 8.71 0.9 H2O
w
xq
961.06 MqH
C51H60 N7O10
C52 H62 N7O10
P
P
w
xq
977.1 MqH
w
1125.0 MqH
xq
xq
6g
6h
C61H70 N7O7 P
C58 H74 N7O13
w
1109.8 MqH
P

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6
D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
Fig. 3.
X
Ž
.
Ž
.
Ž
.
Ž
butyliodide 2a , heptyliodide 2b , decyliodide 2c and
2 -O-ether linkages were formed in the given yields Table
Ž
.
.
1 .
hexadecyliodide 2d . The alkylation reagents for the
oxyethylene modifications were prepared from mono-, di-
Synthesis of the 2X-O-modified adenosine building
Ž
.
or triethylene glycol monomethyl ethers 1e–1g . With the
pentaethylene glycol modification only the diol was avail-
able. Therefore pentaethylene glycol was deprotonated with
NaHrDMF and alkylated using 1.2 eq methyliodide to
blocks was followed by standard protection procedures
established for automated ODN synthesis. First the exo-
cyclic amino function was protected in order to avoid side
reactions. Both the phenoxyacetyl and the benzoyl group
were used for this purpose applying the method of tran-
Ž
.
form the corresponding monomethyl ether 1h in 59%
yield. The dimethyl ether obtained as a side product in an
extent of 5.5% was easily removed by vacuum-flash-chro-
Ž
.
sient protection Ti et al., 1982 . For protection of the
5X-hydroxy function the di-p-methoxytrityl group was used
Ž
.
Ž
.
Ž
.
matography Fig. 2 .
Wagner et al., 1991 . Phosphitylation Table 2 of the
protected alkylated nucleosides was accomplished as de-
The monomethyl ethers were tosylated and converted to
the iodides by a Finkelstein reaction. The synthesis of the
2X-O-alkyladenosines followed the same procedures as out-
Ž
.
scribed Sinha et al., 1984 . The reaction products were
purified by chromatography. Control of purity of these
nucleoside phosphoramidites included use of 31 P-NMR.
Ž
.
lined earlier Wagner et al., 1991 . After selective deproto-
nation of the 2X-hydroxy group of adenosine by use of
NaHrDMF and addition of the alkylation reagents the
Ž .
The phosphoramidite 6i bearing the trifluoracetamide
protected 2X-O-aminohexyl modification
was synthe-
Ž .
i
Fig. 4.

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D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
7
Table 3
Modified dodecamers
5X-AAA AAA AAA AAA-3X
5X-AAA AAA AAA AAA-3X
5X-AAA AAA AAA AAA-3X
5X-AAA AAA AAA AAA-3X
5X-AAA AAA AAA AAA-3X
5X-AAA AAA AAA AAA-3X
Ž
Ž
Ž
Ž
Ž
Ž
.
.
.
7a–i
8a–i
9a–i
.
.
.
10a–i
11a–i
12a–i
exchange chromatography. ODNs containing 2X-O-decyl
Ž
.
and hexadecyl adenosine 7c,d–12c,d proved to be too
lipophilic to be separated by reversed-phase HPLC. ODNs
with these modifications were purified by anion exchange
chromatography performed on a DEAE-material with a
Fig. 5. Retention times of the test oligonucleotides 7a–b,e–i to 12a–b,e–i
referring to an unmodified A12. 7c–d to 12c–d were not included, they
were too lipophilic to be analyzed via RP-HPLC.
Ž
.
NaCl-gradient Rockstroh et al., 1988 . The integrity of
ODNs was verified by MALDI-TOF mass spectra indicat-
ing that modified adenosines had been successfully incor-
porated into the oligomers.
Ž
sized as described earlier Brunar, 1993; Noe and Brunar,
.
1997 .
2.2. Synthesis of modified oligonucleotides
2.3. HPLC-characterization
The phosphoramidites 6a–6i were oligomerized using
an automated DNA synthesizer applying standard 2-
cyanoethyl phosphoramidite chemistry with extended cou-
pling times. To evaluate the influence of the modifications
on the properties of oligonucleotides a series of test dode-
Before purification all modified ODNs were analyzed
by reversed-phase HPLC using the same gradient program.
Retention times were taken as indicators on the lipophilic-
ity of the different sequences.
Retention times were consistent with full length
oligomers. As expected, Fig. 5 shows prolonged retention
times with increasing number of introduced modified 2X-
O-adenosines. It also demonstrates the differences in
lipophilicity between alkyl and oxyethylene modifications.
The change from alkyl chain to the corresponding oxyethy-
lene chain led to a dramatic increase in hydrophilicity.
camers was synthesized in a pattern designed earlier
X
Ž
.
Brunar et al., 1994 . They bore modifications at the 5 -end
in an increasing number up to six modified nucleotides.
Position and extent of substitution of natural nucleotides in
oligonucleotide dodecamers are shown in Table 3.
Coupling yields for incorporation of 2X-O-alkyladeno-
sine varied between 70 and 99%. All oligonucleotides
were purified by trityl off reversed-phase HPLC or anion
Ž
Fig. 6. CD-spectra of duplexes formed upon hybridization of T12 with pentaethyleneglycol modified dA12 7h–12h 08C; 9 mM; 0.15 M NaCl, 0.01 M
.
Tris–HCl; pH 7 .

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D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
Ž
Fig. 7. CD-spectra of duplexes formed upon hybridization of T12 with hexadecyl modified dA12 7d–10d 08C; 9 mM; 0.15 M NaCl, 0.01 M Tris–HCl;
.
pH 7 .
2.4. CD-spectra of RNA–DNA hybrids
a flattening out of the curves was detectable. This effect
was more distinct in the alkyl series than the oxyethylene
series. However, a complete switch of double helix confor-
mation of a 2X-O-modified RNA–DNA hybrid duplexes
into an A form could not be found, regardless of the extent
of modification. CD peaks of the hybrid duplexes both at
220 nm and 265 nm, bore in all cases resemblance to the B
form shape of the dA12-T12 duplex.
A main point of interest was the change of duplex
geometry and stability caused by different modifications
and different extent of modification. Generally, a transition
from B to A form occurs within a mixed hybrid, when a
Ž
.
DNA–DNA hybrid segment B-form transforms to a
Ž
. Ž
.
RNA–DNA hybrid A-form Wang et al., 1982 . It is also
known that a hybrid duplex containing purine-rich RNA
segments adopts a conformation with more A-form simi-
2.5. Melting behaÕior
Ž
.
larities than B-form similarities Ratmeyer et al., 1994 . To
investigate the duplex geometry of these dodecamers, they
were hybridized to an unmodified T12 strand.
Measurement of the melting temperatures is a common
method to determine duplex stability. In this investigation,
duplex stabilities were measured by recording temperature
dependences of the CD signal at 250 nm in a temperature
range from 0–708C. Oligonucleotide concentrations were 9
In Figs. 6 and 7 selected examples of CD-spectra are
given. All spectra of the modified ODNs took characteris-
tic B-form shapes. With increasing extent of modifications
Fig. 8. Melting temperature versus strand substitution in 2X-O-alkyl series
Fig. 9. Melting temperature versus strand substitution in 2X-O-oxyethylene
Ž
.
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž . Ž . Ž . Ž .
. Ž .
diamond modified ODNs. Concentration each strand is 9 mM.
with i circle , a square , b up triangle , c down triangle , d diamond
series with i circle , e square , f up triangle , g down triangle , h
Ž
Ž
modified ODNs. Concentration each strand is 9 mM.

(
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D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
9
mM for each strand. All modified oligonucleotide dode-
camers were hybridized to T12.
Sequences 7i–12i exhibited melting temperatures which
were higher than those of all other modifications. This
supports the assumption of a stabilizing effect of amino-
hexyl modifications and this effect exceeded that of all
other shown modifications. Taking into account that the
repulsion of the negatively charged backbones of the com-
plementary strands is a destabilization factor in double
helix formation, melting temperatures of 7i–12i support
the concept of duplex stabilization through charge neutral-
ization by introduction of zwitterionic sites. Whether an
additional stabilizing effect is exerted by interstrand salt
formations between phosphate anions and ammonium
cations, as suggested by molecule dynamics simulations,
can not be decided at present.
Fig. 10. The calculated DG of the modifications 7a–7i including an
unmodified A12–T12.
indicated stabilization forces of the oxyethylene moieties.
This effect had a minimum at the 7f modification. The DG
value of 7i was similar to the modifications of correspond-
ing chainlength 7b and 7f.
Not surprisingly, a comparison between the alkyl series
Ž
.
Ž
.
Fig. 8 and the oxyethylene series Fig. 9 showed lower
melting temperatures and thus lower duplex stabilities of
the alkyl series. Unpredictable however, was the result that
ODNs with only one modified oxyethylene nucleotide
7e,g,h exhibited higher melting temperatures when com-
3. Conclusion
Ž
Ž
..
pared to unmodified A12–T12 34.88C 9 mM , while
upon introduction of further modified units an expected
slight decrease in duplex stability was observed.
By its ability to reduce the net charge of the duplex, the
aminohexyl modification lead to duplex stabilization. Also,
oxyethylene modifications showed stabilization properties
in case of introduction of only one moiety. However, in all
cases an increase of extent of modification resulted in
melting temperatures below the unmodified A12–T12 du-
plex. The decrease in melting temperatures took the fol-
lowing order:
2.6. Thermodynamic data of 2X-O-modified sequences
Duplex free energy values were determined from melt-
ing curves by plotting 1rTm versus the natural logarithm
of the total oligonucleotide concentration. Linear regres-
Ž
.
sion leads to determination of D H, DS and DG Table 4
Ž
.
Marky and Breslauer, 1987 .
aminohexyl)oxyethylene)alkyl modifications.
In the alkyl series 7a–c the data exhibited a decrease
The steric hindrance exerted by a bulky alkyl 2X-O-
moiety was compensated by the neutralizing effect gener-
ated by introduction of an amino group. Also, oxyethylene
chains which might exert positive effects in therapeutic
applications due to their amphiphilic character, exhibited
in all cases increased duplex stability when compared to
the corresponding alkyl modifications. These results indi-
cate strongly that the introduction of zwitterionic modifica-
tions or oxyethylene chains into the 2X-O-position might
provide useful tools for the modulation of properties of
antisense oligonucleotides in their development as drugs.
with increasing chainlength Fig. 10. The DG of 7d showed
a value similar to 7a. In the oxyethylene series, 7e,g,h
showed values higher than the unmodified A12–T12. This
Table 4
Thermodynamic data
y1
y1
y1
Ž
.
Ž . Ž
.
Modification D H kJ mol
DS J K moly1 DG kJ mol
a
b
c
d
e
f
g
h
i
y251.42
y162.80
y250.28
y276.5
y361.18
y133.84
y308.06
y255.99
y328.89
y713.74
y425.69
y740.96
y798.57
y1064.25
y328.73
y892.03
y717.36
y996.87
y929.45
y38.62
y35.88
y29.37
y38.41
y43.87
y35.83
y42.1
4. Experimental protocols
y42.1
y31.68
y41.81
4.1. Chemistry
Unmodified y318.92
For details of the chemical synthesis of the phospho-
The calculations have been accomplished by choosing T s298.15 K
Ž
.
ramidites 6a–6h see Werner and Noe 1997 . For details
Ž
.
Ž
according to literature Marky and Breslauer, 1987 . Values are kJ
moly1
.
Ž
.
.
of the phosphoramidite 7i see Brunar et al. 1997 .

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10
D. Werner et al.rPharmaceutica Acta HelÕetiae 73 1998 3–10
Wirksamkeit von funktionalisierten Oligonukleotiden. Dissertation,
4.2. HPLC-analyses
Wien.
Brunar, H., Haberhauer, G., Werner, D., Noe, C.R., 1994. 2X-O-Modified
Oligonucleotides: Synthesis and biophysical analysis. Eur. J. Pharma-
ceut. Sci. 2, 150.
Solutions of crude oligonucleotides were filtered on
Schleicher and Schuell Spartan 13r20, 0.45 mm Braun-
rand H filters, concentrated via speed vac and redissolved
Brunar, H., Werner, D., Haberhauer, G., Gilbert, M., Noe, C.R., 1997.
Nucleic Acids: Synthesis and Base Pairing Properties of 2X-Aminoal-
kyl Homopurin RNA. In preparation.
Cotten, M., Oberhauser, B., Brunar, H., Holzner, A., Issakides, G., Noe,
C.R., Schaffer, G., Wagner, E., Birnstiel, M.L., 1991. 2X-O-methyl,
2X-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyri-
bonucleotides as inhibitors of the in vitro U7 snRNP-dependent
mRNA processing event. Nucleic Acids Res. 19, 2629–2635.
Crooke, S.T., 1995. Delivery of oligonucleotides and polynucleotides. J.
Drug Targeting 3, 185–190.
Ž
.
in 300 ml H2O MilliQ for determination of oligonucleo-
Ž
.
tide concentrations by UV measurement 260 nm . For
preparative HPLC the volume of oligonucleotide contain-
ing solution was reduced under 1000 ml and injected to
reverse phase VarioPrep ET 250r10 Nucleosilw 100-7
Ž
.
C18 Macherey Nagel . A solvent gradient of triethylam-
Ž
. Ž .
monium acetate 100 mM, pH 7.0 A and 80% CH3CN
. Ž .
Ž
in triethylammonium acetate 100 mM, pH 7.0 B was
Englisch, U., Gauss, D.H., 1991. Chemisch modifizierte Oligonucleotide
als Sonden und Agentien. Angew. Chem. 103, 629–739.
Ž
.
used as mobile phase flow rate 16 mlrmin; 30–50 psi .
Purified oligonucleotides from preparative HPLC were
Ž
.
Erickson, R.P., Izant, J.G. Eds. , 1992. Gene Regulation: Biology of
Antisense RNA and DNA. Raven Press, Ltd., New York.
Lesnik, E.A., Guinosso, C.J., Kawasaki, A.M., Sasmor, H., Zounes, M.,
Cummins, L., Ecker, D.J., Cook, P.D., Freier, S.M., 1993.
Oligodeoxynucleotides Containing 2X-O-Modified Adenosine: Synthe-
sis and effects on stability of DNA:RNA Duplexes. Biochemistry 32,
7832–7838.
Ž
.
lyophilized and redissolved in 500 ml H2O MilliQ ,
desalted on Waters SEP-PAKw columns, concentrated via
Ž
.
speed vac and redissolved in 300 ml H2O MilliQ . Por-
Ž
.
tions of 20 ml 0.2 mgrml were injected to a ET 250r4
Nucleosilw 100-5 C18 Macherey Nagel column for ana-
Ž
.
Marky, L.A., Breslauer, K.J., 1987. Calculating thermodynamic data for
transitions of any molecularity from equilibrium melting curves.
Biopolymers 26, 1601–1620.
Ž
.
lytical HPLC flow rate 1 mlrmin; 150–160 psi .
Martin, P., 1995. Ein neuer Zugang zu 2X-O-Alkylribonucleosiden und
Eigenschaften deren Oligonucleotide. Helv. Chim. Acta 78, 486–504.
Noe, C.R., Brunar, H., 1997. Modifizierte Oligonukleotide, Verfahren zu
ihrer Herstellung und Verwendung als Wirkstoffe, PCT Int. Appl.
4.3. CD-spectroscopy
Concentrations of purified and desalted oligonucleotides
were determined by UV measurement at 260 nm using
molar extinction coefficients of 15,400 cmy1 My1 for the
dA-, 2X-O-alkyl A residues and 8800 cmy1 My1 for the T
residues. Aliquots of purine and pyrimidine oligonucleo-
tides were transferred into microcentrifuge tubes mixed
with 30 ml of a 1.5 M NaCl, 0.1 M Tris–HCl buffer at pH
7.0 and enough H2O MilliQ to a total volume of 300 ml.
The oligonucleotide solutions were allowed to hybridize at
608C for 3 min, then cooled to RT and transferred into a
0.1 cm pathlength cuvette. CD-spectra of the oligonucleo-
tide duplexes were obtained at 108C in a wavelength range
of 320 to 210 nm. Temperature dependent measurement of
ellipticity was performed from 0–608C with a temperature
slope of 508C per hour. A range of oligomer strand
concentrations and a 1:1 ratio of the two complementary
strands were used to allow calculations of the thermody-
namic parameter for duplex formation. Parameters were
¨
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30273.
Ratmeyer, L., Vinayak, R., Zhong, Y., Zon, G., Wilson, W.D., 1994.
Sequence specific thermodynamic and structural properties for DNA-
RNA duplexes. Biochemistry 33, 5298–5304.
Reddy, D.S., 1996. Antisense oligonucleotides: A new class of potential
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anti-AIDS and anticancer drugs. Drugs Today 32 2 , 113–137.
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Rockstroh, H., Wangler, W., Ackermann, Th., 1988. Preparative fraction-
ation of oligoriboguanylates by anion-exchange chromatography on
TSK DEAE-5PW. J. Chromatogr. 442, 401–406.
Sinha, N.D., Biernat, J., McManus, J., Koster, H., 1984. Polymer support
¨
oligonucleotide synthesis XVIII: Use of b-cyanoethyl-N,N-dial-
kylamino-rN-morpholino phosphoramidite of deoxynucleosides for
the synthesis of DNA fragments simplifying deprotection and isola-
tion. Nucleic Acids Res. 12, 4539–4557.
Sproat, B.S., Iribarren, A.M., Beijer, B., Pieles, U., 1991. 2X-O-Al-
kyloligoribonucleotides: Synthesis and applications in studying RNA
splicing. Nucleosides Nucleotides 10, 25–36.
Ti, G.S., Gaffney, B.L., Jones, R.A., 1982. Transient protection: Efficient
one-flask synthesis of protected deoxynucleotides. J. Am. Chem. Soc.
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Ž
determined by the van’t Hoff method Marky and Bres-
.
lauer, 1987 .
Wagner, E., Oberhauser, B., Holzner, A., Brunar, H., Issakides, G.,
Schaffner, G., Cotten, M., Knollmuller, M., Noe, C.R., 1991. A
¨
simple procedure for the preparation of protected 2X-O-methyl or
2X-O-ethyl ribonucleoside-3X-O-phosphoramidites. Nucleic Acids Res.
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