ABSTRACT
We have designed and synthesized a series of novel antisense methylphosphonate
oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non-nucleotide-based linking moiety near the middle of the sequence in place
of one of the nucleotide bases. The region surrounding the unpaired base on the
RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by
ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising
hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting
acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered
to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.
Antisense oligonucleotides have received considerable attention for their
ability to modulate gene expression. A wide variety of backbone modifications
have been developed with the intention of providing metabolically stable
oligonucleotide analogs for
in vivo
applications. Yet, with the exception of the phosphorothioates and
phosphorodithioates, none of these analogs has been found to activate
ribonuclease H (RNase H), an endogenous enzyme that cleaves mRNA at the site of
a DNA/RNA heteroduplex (
1
,
2
). This has proved to be a disappointment, since recent studies have shown that
unmodified oligonucleotides act predominantly through an RNase H cleavage mode
(
3
-
5
). One way to remedy this deficiency is to insert short stretches of
phosphodiester or phosphorothioate DNA into an otherwise nuclease-stable backbone (
6
,
7
). Alternatively, a number of investigators have focused on the development of
synthetic ribonuclease mimics (artificial ribonucleases) that can be covalently
attached to an oligonucleotide and thereby promote a cleavage reaction on its
RNA target (
8
-
15
).
One of the apparent shortcomings of previously described artificial
ribonucleases is that they are unable to bind and distort the RNA strand into a
preferred geometry for cleavage. For example, bovine RNase A binds RNA-like substrates in a non-helical conformation wherein the bases on either side of the bond
that is cleaved point away from each other (
16
). Helical RNA is known to be resistant to hydrolytic cleavage compared with
single-stranded RNA. This appears to be an intrinsic property of the 3'-5' linkage, which has been shown to be ~900 times more resistant to hydrolysis in double-stranded RNA compared with a 2'-5' linkage (
17
). Considering such evidence, we reasoned that the base stacking that occurs
within and around an antisense heteroduplex would render the RNA strand less
susceptible to cleavage by artificial nucleases. Further, we assumed that the
rotation of a single base on the RNA strand outward from a stacked helical
geometry would position its 2'-hydroxyl in a spatial orientation analogous to the 3'-hydroxyl in a duplex containing a 2'-5' linkage (cited above). Therefore,
we considered replacing one of the bases in the antisense strand with a non-complementary, non-nucleotide-based linker (
18
).
Here we describe a heteroduplex motif employing a non-complementary, non-nucleotide-based linker within an antisense methylphosphonate oligonucleotide (MPO) strand that
accelerates the rate of hydrolytic cleavage on its RNA target in a site-specific manner. We also report that certain simple catalytic moieties
covalently linked to the antisense strand are capable of cleaving the RNA
strand in this motif.
1
H NMR spectra were obtained on a 300 MHz Bruker ARX 300 Spectrometer. All
1
H NMR results were obtained in CDCl
3
unless otherwise indicated. FTIR spectra were recorded on a Mattson Galaxy
Series FTIR 3000 spectrometer. Mass spectra were recorded on a Fisons Trio 2000
Spectrometer and provided by JBL Scientific (San Luis Obispo, CA). Melting
points are uncorrected.
t-Butyl pent-4-enoate (
1
).
A solution of 4-pentenoic acid (10 g) in t-butyl acetate (120 ml) was treated with perchloric acid (4 drops)
and stirred at room temperature for 16 h. The reaction was quenched by addition
of aqueous saturated sodium bicarbonate (100 ml) and stirred for 15 min. The
phases were separated and the organic phase was further washed with sodium
bicarbonate solution, dried over anhydrous MgSO
4
, filtered and concentrated. The resulting crude product was purified by vacuum
distillation: b.p. 51-55oC/20 torr (lit. 51-53oC/20 torr;
19
); yield 6.4 g.
t-Butyl 4,5-dihydroxypentanoate (
2
).
To a solution of
N
-methylmorpholine oxide (2.50 g, 1.1 eq.), acetone (3 ml) and water (8 ml)
was added a solution of osmium tetroxide (20 mg) in t-butanol (1.5 ml) followed by ester
1
(3.00 g, 1 eq.). The heterogeneous reaction mixture was stirred at room
temperature for 16 h. A slurry of sodium hydrosulfite (330 mg), magnesium
silicate (3.0 g) and water (25 ml) was prepared and added to the reaction
mixture and stirred for 5 min. The reaction mixture was filtered through celite
and the filtrate was extracted with ethyl acetate (2 * 50 ml). The combined organic extracts were dried (MgSO
4
), filtered and concentrated to give a clear viscous oil (yield 2.95 g, 80%).
1
H NMR: [delta] 2.41 (t, 2H), 1.45 (s, 9H); IR, 1728 (s), 3402 (br m) cm
-1
; MS(ESI,
m
/
z
), 191 (theor. 190.24).
t-Butyl 4,5-bis-tosyloxypentanoate (
3
).
A solution of the diol
2
(1.0 g) and triethylamine (3.0 ml) in dichloromethane (20 ml) was cooled to 0oC and then added dropwise to a solution of tosyl chloride (2.10 g) in
dichloromethane (15 ml) at 0oC. Following this addition the reaction mixture was stirred at 0oC for 30 min and then at room temperature for 16 h. The solvent was
removed and the residue was treated with ethyl acetate (50 ml) and water (35
ml). The organic phase was separated and washed with saturated solutions of
sodium bicarbonate and brine (30 ml each), dried (MgSO
4
), filtered and concentrated to give a colorless oil (2.92 g). Chromatographic
purification on silica gel using hexane/ethyl acetate (3:1) eluent yielded 1.76
g (67% yield) of the ditosylate
3
.
1
H NMR: [delta] 4.66 (m, 1H), 2.17 (dt, 2H), 1.83 (dt, 2H), 1.35 (s, 9H); MS(ESI,
m
/
z
), 499 (theor. 498.61).
t-Butyl 4,5-diazidopentanoate (
4
).
To a solution of the ditosylate
3
(3.5 g, 1 eq.) in dimethylformamide (20 ml) was added sodium azide (0.936 g,
2.05 eq.). The reaction mixture was heated at 60oC for 16 h. Next, the reaction was cooled to room temperature and the
solvent was removed under reduced pressure. The residue was partitioned between
ethyl acetate (30 ml) and water (20 ml) and the organic phase was separated and
washed further with water (30 ml), dried (MgSO
4
), filtered and concentrated to give 1.5 g (88% crude yield) of the diazide
4
as a colorless oil.
1
H NMR: [delta] 3.60 (m, 1H), 3.45 (m, 2H), 1.8 (m, 2H), 1.46 (s, 9H); IR, 2104 (s),
1727 (m).
t-Butyl 4,5-diaminopentanoate (
5
).
A solution of the diazide
4
(380 mg) in methanol (3 ml) was added to a pre-hydrogenated mixture of 10% palladium on carbon (20 mg) in methanol (5
ml). The mixture was placed under an atmosphere of hydrogen and stirred at room
temperature for 3 h. The catalyst was removed by filtration through celite and
the filtrate was concentrated to give 282 mg of
5
(97% crude yield) as a colorless oil which was used without further
purification.
1
H NMR (CDCl
3
): [delta] 2.3 (m, 2H), 1.43 (s, 9H); IR, 1721 (s), 3300 (br m).
t-Butyl 4,5-bis(fluorenylmethoxycarbamato)-diaminopentanoate (
6
).
To a solution of the diamine
5
(50 mg, 1 eq.) in dichloromethane (2 ml) was added a solution of
fluorenylmethoxycarbonyl suberimidate (188 mg, 2.1 eq.) in dichloromethane (2
ml). The reaction was stirred at room temperature for 16 h. Then the solvent
was removed under reduced pressure and the residue was partitioned between
ethyl acetate (10 ml) and water (10 ml). The organic phase was dried (MgSO
4
), filtered and concentrated to give 260 mg of a white solid. The desired
biscarbamate
6
was purified by flash chromatography on silica gel using hexanes/ethyl acetate
(2:1) as eluent. The final yield was 120 mg of a white crystalline solid (71%
yield).
1
H NMR: [delta] 3.7 (m, 1H), 3.3 (br t, 2H), 2.3 (t, 2H), 1.43 (s, 9H).
4,5-Bis(fluorenylmethoxycarbonamato)-diaminopentanoic acid (
7
).
The ester
6
(60 mg) was treated with trifluoroacetic acid/dichloromethane (1:1, 4 ml)
solution. The reaction was stirred at room temperature for 3 h. The solvents
were then removed under reduced pressure and the residue was treated with ethyl
acetate (4 ml). The resulting slurry was then placed in the freezer overnight
and the precipitate was recovered by filtration and dried. Yield 35 mg (64%).
1
H NMR: [delta] 3.7 (m, 1H), 2.3 (t, 2H); MS(ESI,
m
/
z
) 577.5 (theor. 576.65).
Succinimidyl 4,5-bis(fluorenylmethoxycarbonamato)-diaminopentanoate (
8
).
To a solution of the acid
7
(20 mg, 1 eq.) in dioxane (1 ml) and triethylamine (5.6 [mu]l, 1.1 eq.) was added dropwise a solution of succinimidyl 1,2,2,2-tetrachloroethylcarbonate (11 mg, 1 eq.) in dioxane (1 ml). The
reaction mixture was stirred at room temperature for 3 h. Then the mixture was
concentrated to dryness under reduced pressure. The residue was dissolved in
ethyl acetate (10 ml) and washed with 10% aqueous citric acid and saturated
aqueous sodium bicarbonate and brine (10 ml each), dried (MgSO
4
), filtered and concentrated to give a white solid (25 mg). The product
8
was recrystalized from hexanes/ethyl acetate to give a white crystalline solid
(16 mg, 68% yield).
1
H NMR (CDCl
3
): [delta] 3.76 (m, 1H), 3.30 (t, 2H), 2.82 (s, 4H), 2.68 (t, 2H); MS(ESI,
m
/
z
) 674.4 (theor. 673.7).
Methyl 3,5-bis(isothiocyanatomethyl)benzoate (
11
).
To a solution of methyl 3,5-bis(bromomethyl)benzoate (1.0 g, 1 eq.) in dimethylformamide (20 ml) was
added sodium azide (0.4 g, 2 eq.). The reaction mixture was heated in an oil
bath to 70oC for 15 min and then allowed to cool to room temperature. Then the mixture
was concentrated under reduced pressure and the residue was partitioned between
ethyl acetate and water. The organic phase was washed three times with water,
once with brine, dried (MgSO
4
), filtered and concentrated under reduced pressure to give methyl 3,5-bis(azidomethyl)benzoate (
9
) as a yellow oil. The crude product was used without further purification.
R
f
0.42 (4:1 hexane/ethyl acetate); IR (thin film), 2953, 2102, 1724, 1435, 1307,
1224 cm
-1
;
1
H NMR: [delta] 7.97 (s, 2H), 7.49 (s, 1H), 4.45 (s, 4H), 3.95 (s, 3H). A stirred
suspension of
9
(0.77 g) and 10% palladium on carbon (0.07 g) in methanol (5 ml) was degassed
at room temperature and then subjected to 1 atm hydrogen gas via inflated
balloon for 12 h. Excess hydrogen was removed under vacuum and the reaction
vessel was purged with argon. The reaction mixture was filtered through Celatom
FW-14 with methanol washings and the filtrate was concentrated under reduced
pressure to give methyl 3,5-bis(aminomethyl)benzoate (
10
) as a pale yellow oil (yield 0.60 g). The crude product was used without
further purification. IR (thin film), 3357, 3296, 2951, 1721, 1651, 1602, 1544,
1435, 1307, 1224 cm
-1
;
1
H NMR (DMSO-d
6
): [delta] 7.76 (s, 2H), 7.56 (s, 1H), 3.85 (s, 4H), 3.79 (s, 3H). To a stirred
suspension of
10
in a 1:1 mixture of toluene/water (20 ml) was added sodium carbonate (0.83 g,
7.8 mmol). The mixture was cooled in an ice/water bath and then thiophosgene
(0.52 ml, 6.86 mmol) was added dropwise by syringe. The reaction was warmed to
room temperature and allowed to stand overnight. The resulting bilayer was
extracted with ethyl acetate. The organic extracts were washed with saturated
aqueous sodium bicarbonate and brine, dried (MgSO
4
), filtered and concentrated under reduced pressure to give a yellow oil.
Purification by flash chromatography on silica gel using 9:1 hexane/ethyl
acetate followed by 7:3 hexane/ethyl acetate as eluents gave 0.39 g of the
desired product
11
as a thick oil, which solidified upon standing in a refrigerator (43% yield
after three steps). m.p. 67-69oC; IR (thin film), 2950, 2185, 2096, 1722, 1609, 1434, 1305, 1222 cm
-1
;
1
H NMR: [delta] 7.95 (s, 2H), 7.48 (s, 1H), 4.81 (s, 4H), 3.93 (s, 3H).
Succinimidyl 3,5-bis-[N
'
-(tert-butoxycarbonyl)histidyl]-N-thioureamidylmethyl benzoate (
14
).
To a solution of
11
(0.20 g, 1 eq.) in tetrahydrofuran (4 ml) was added histamine dihydrochloride
(0.26 g, 1 eq.) and 1 M sodium carbonate solution (1.4 ml, 2 eq.). The
resulting biphasic mixture was heated at 45oC for 5 h. The reaction mixture was transferred to a separating funnel and
the organic phase was separated and concentrated under reduced pressure to give
a wet oily residue. The crude residue was dissolved in methanol (3 ml) and then
treated with 1 M sodium hydroxide solution (1.1 ml, 1.1 eq.). The reaction
mixture was heated at 40oC for 4 h and then concentrated to give the sodium carboxylate intermediate
(
12
) as an oily residue. This material was dissolved in dioxane (3 ml) and treated
with di-t-butyldicarbonate (0.47 g, 2.16 mmol). The reaction was stirred at
room temperature for 12 h, diluted with water (5 ml) and extracted with ethyl
acetate. The aqueous phase was acidified to pH 3 with 3 M aqueous HCl and then
extracted with ethyl acetate. The organic phase was washed with brine, dried
(MgSO
4
), filtered and concentrated under reduced pressure to give 0.20 g t-butylcarbonyl-protected intermediate (
13
) as a crude white solid.
1
H NMR (acetone-d
6
): [delta] 8.00 (s, 2H), 7.93 (s, 1H), 7.54 (br s, 2H), 7.26 (s, 2H), 4.82 (br s,
4H), 3.79 (br m, 4 H), 2.80 (t,
J
= 6.8 Hz, 4H), 1.6 (s, 18H). To a solution of
13
(0.20 g) in dichloromethane (4 ml) were added
N
,
N
'-dicyclohexylcarbodiimide (0.069 g, 0.33 mmol) and
N
-hydroxysuccinimide (0.038 g, 0.33 mmol). The reaction mixture was stirred
at room temperature for 4 h and then filtered to remove the urea by-product. The filtrate was concentrated under reduced pressure to give a
semi-solid residue. Purification by flash chromatography on Fluorisil using
24:1 dichloromethane/isopropanol as eluent gave 49 mg of the desired product (
14
) as a white solid (9% yield after 4 steps). m.p. 100oC (dec.);
1
H NMR: [delta] 7.95, (s, 2 H), 7.90 (s, 2H), 7.59, (s, 1H), 7.14 (s, 2H), 4.74 (br m,
4H), 3.67 (br m, 4 H), 2.90 (s, 4H), 2.72 (t,
J
= 6.0 Hz, 4H), 1.60 (s, 18H); MS(ESI,
m
/
z
), 784.4 (theor. 783.93).
N-
[epsilon]
-Fluorenylmethoxycarbonyl-L-lysyl-N-
[alpha]
-fluorenylmethoxycarbonyl-N-im-trityl-histidine (
15
).
To a solution of
N
-[epsilon]-fluorenylmethoxycarbonyl-L-lysine (100 mg, 0.37 mmol) (Bachem) in
dichloromethane (5 ml) was added
N
,
N
-diisopropylethylamine (0.81 mmole, 2.2 eq.) and chlorotrimethylsilane
(0.41 mmol, 1.1 eq.) dropwise with stirring. The reaction mixture was stirred
for 0.5 h at room temperature. Next,
N
-[alpha]-fluorenylmethoxycarbonyl-
N
-im-trityl-histidine pentafluorophenyl ester (250 mg, 0.9 eq.)
(Novabiochem) was added and the mixture was stirred for an additional 1 h at
room temperature. The solvent was then removed under reduced pressure and the
product was dissolved in dichloromethane (2 ml) and purified by flash column
chromatography (1 * 30 cm) on silica gel (230-400 mesh) using 2% methanol in dichloromethane as the eluent. The
transient silyl protecting group was removed during this process. The yield of
the product was 250 mg (89%). MS(ESI,
m
/
z
), 970 (theor. 970);
1
H NMR (CDCl
3
): [delta] 1.49 (m, 6H, CH
2
), 1.89 (m, 2H, CH
2
), 2.68 (m, 1H, CH), 3.06 (m, 2H, CH
2
), 4.04-4.20 (5H), 4.35 (m, 1H, =CH), 4.57 (m, 1H, =CH), 4.88 (m, 2H), 7.02-7.74 (31 aromatic Fmoc and trityl protons).
N-
[epsilon]
-Fluorenylmethoxycarbonyl-L-lysyl-N-
[alpha]
-fluorenylmethoxycarbonyl-N-im-trityl-histidine pentafluorophenyl ester (
16
).
Compound
15
(200 mg) was dissolved in dimethylformamide (1 ml) and pyridine (2 eq.) was
added. Pentafluorophenyl trifluoroacetate (1.3 eq.) was added and the reaction
mixture was stirred at room temprature for 1 h. The progress of the reaction
was monitored by TLC on silica gel (1:1 ethyl acetate/hexane). Then the
reaction mixture was diluted with ethyl acetate (100 ml) and extracted with 0.1
M aqueous HCl (2 * 100 ml) followed by 5% sodium bicarbonate (2 * 100 ml). The organic phase was separated, dried (MgSO
4
) and evaporated to dryness under reduced pressure. The yield of the product was
150 mg, which was estimated to be >90% pure by TLC and was used without further
purification. MS(ESI,
m
/
z
), 1136 (theor. 1136);
1
H NMR (CDCl
3
): [delta] 1.55 (m, 4H, CH
2
), 2.01 (m, 2H), 2.90 (m, 1H, CH), 3.22 (m, 2H, CH
2
), 4.08-4.35 (3H), 7.02-7.74 (31 H, aromatic Fmoc and trityl protons).
Synthetic RNA was prepared as described previously (
20
). The non-nucleotide based, non-complementary linking reagent, having the parent structure shown in
Figure
1
, has also been described (
18
). This reagent was coupled into the methylphosphonate oligonucleotide (MPO) at
the indicated position within the sequence using an automated DNA synthesizer (
18
,
21
-
22
).
Compounds
8
,
14
and
16
were conjugated onto a methylphosphonate oligonucleotide having a non-nucleotide, non-complementary amino linker inserted in the middle of the sequence (G-1719) to give oligonucleotide conjugates G-2337, G-2437 and G-2435 respectively.
Conjugation reactions.
Approximately 60 A
260
U methylphosphonate oligonucleotide G-1719 were dissolved in 1:1 acetonitrile/water (100 [mu]l). Next, the following reagents were added in the order indicated,
with mixing after each addition to avoid precipitation: dimethylsulfoxide
(DMSO, 215 [mu]l), 1 M aqueous HEPES buffer, pH 8.0 (50 [mu]l) and a 100 mM solution of either
8
,
14
or
16
in anhydrous DMSO (35 [mu]l). The resulting mixture was reacted at room temperature for 1 h. At the
end of this time period additional aliquots of 100 mM
8
,
14
or
16
(in DMSO, 12.5 [mu]l), DMSO (12.5 [mu]l), 1 M HEPES (pH 8.0, 5 [mu]l), and sterile water (20 [mu]l) were added. After mixing the reaction was allowed to continue
for an additional 1 h. Absolute ethanol (1 ml) was added and the tube was
chilled at -20oC to precipitate the conjugated product oligonucleotide. Then the
tube was centrifuged for 15 min in a microcentrifuge at 4oC. The supernatant was discarded and the resulting pellet was resuspended
in 1:1 acetonitrile/water (400 [mu]l).
HPLC analysis and purification.
The crude protected oligonucleotide conjugates described above were purified by
reversed phase HPLC chromatography using a Beckman System Gold HPLC system
equipped with a Model 168 diode array detector. A Hamilton PRP-1 polymeric reversed phase column was used (4.1 * 250 mm). Solvent A, 100 mM ammonium acetate (pH 6); solvent B;
acetonitrile. Program 1 (for compounds 2337-1 and 2347-1): 0-15% solvent B (0-8 min), 15-45% solvent B (8-38 min), flow rate 1.5 ml/min; program 2
(for compound 2435-1): 0-15% solvent B (0-8 min), 15-70% solvent B (8-60 min), flow rate 1.5 ml/min. Retention times
for the protected forms of the conjugated oligonucleotides were as follows: G-2337 (protected), 32.9 min; G-2435 (protected), 40.6 min; G-2437 (protected), 31.2 min. (At this stage Fmoc protecting
groups remained on G-2337 and G-2435, a trityl protecting group remained on G-2435 and a Boc protecting group remained on G-2437.)
Removal of protecting groups.
The HPLC-purified protected forms of oligonucleotide conjugates G-2337, G-2435 and G-2437 from the previous step were dissolved in 47.5%
acetonitrile/47.5% ethanol/5% water (1 ml) and redistilled ethylenediamine (1
ml) was added. After 1 h the reaction mixtures were diluted with water (9 ml)
and neutralized with glacial acetic acid (0.6 ml). These solutions were then
applied to a Sep-Paktm C18 cartridge (Millipore Corp.) that had been previously washed
with acetonitrile (5 ml), 1:1 acetonitrile/water (5 ml) and water (5 ml). After
application of the samples the cartridges were washed with water (8 ml). In the
case of G-2435 the cartridge was also washed with 2% aqueous trifluoroacetic acid (5
ml) to remove the trityl protecting group. The products were then eluted from
the cartridges with 1:1 acetonitrile/water (5 ml) and concentrated to dryness
under reduced pressure. Following removal of the protecting groups the desired
oligonucleotide conjugates were further purified by HPLC as described above.
Retention times for the deprotected oligonucleotide conjugates were as follows:
G-2337, 15.7 min (program 1); G-2435, 23.6 min (program 2); G-2437, 20.8 min (program 1). MS(ESI,
m
/
z
): G-2337, 5407.9 (theor. 5408); G-2435, 5560.5 (theor. 5559.3); G-2437, 5763.9 (theor. 5762.0).
Annealing reaction mixtures contained equimolar amounts of antisense
oligonucleotide analog and RNA target oligonucleotide (2.4 [mu]M total strand concentration), 20 mM potassium phosphate (pH 7.2), 100 mM
sodium chloride, 0.1 mM EDTA and 0.03% potassium sarkosylate. The reaction
mixtures were heated to 80oC, cooled slowly to room temperature over ~2 h and then chilled to 4oC. The annealed samples were then transferred to 1 cm quartz
cuvettes (pre-cooled to 4oC) and placed in a Varian Cary Model 3E spectrophotometer containing
a temperature controlled 6 * 6 sample holder and interfaced to an IBM-compatible PC computer. Denaturation was monitored at 260 nm as a
function of temperature, increasing from 5 to 80oC at a ramp rate of 1oC/min. Melting temperatures (
T
m
) are defined at the point where 50% of the duplex had denatured and have an
error of ~+-1oC. Based on this analysis, G-1719 (i.e. the unconjugated precursor MPO) was found to
have a
T
m
of ~29oC. Each of the conjugated MPOs (G-2337, G-2435 and G-2437) had
T
m
values ranging from 30 to 32oC.
Several compounds were synthesized for conjugation onto an MPO having a primary
non-nucleotide-based alkylamino linker in place of one of the complementary bases
(G-1719, Fig.
1
). Earlier work in our laboratory demonstrated that nucleotide bases on either
side of this linker retain their ability to hydrogen bond and stack in an
MPO/RNA heteroduplex (
18
). We chose to insert this linker in place of one of the complementary bases,
reasoning that the opposing base on the complementary RNA strand would be less
constrained to a stacked, helical geometry, thereby rendering its
phosphodiester bonds more susceptible to hydrolytic cleavage. Each of these
compounds was designed to provide a moiety on the MPO that is capable of
catalyzing a hydrolytic cleavage reaction on its complementary RNA target. To
facilitate the design process molecular modeling studies were conducted to
ensure that the catalytic moieties are capable of spanning the distance from
the antisense MPO strand to the RNA backbone (M.A.Reynolds and T.A.Larsen,
unpublished results; models were created using Insight II software from Biosym
Inc.). Compound
8
is a derivative of ethylenediamine (EDA). EDA is capable of catalyzing the
hydrolytic cleavage of single-stranded RNA around pH 7-8 (M.A.Reynolds, T.A.Beck, P.B.Say, unpublished results;
17
,
23
,
24
). Compound
14
contains two side chains terminating in imidazole moieties. Imidazole, like EDA,
catalyzes the cleavage of single-stranded RNA at neutral pH (
25
,
26
). We assumed that two imidazole moieties tethered to the same molecule would
have a synergistic catalytic effect, as has been demonstrated elsewhere (
27
). Compound
16
was designed based on a screen of various di- and tripeptides for their ability to catalyze RNA hydrolysis
(M.A.Reynolds and P.B.Say, unpublished results).
These compounds were individually coupled onto G-1719 by solution phase coupling chemistries and the products were purified
by HPLC chromatography. The structures of the resulting oligonucleotide
conjugates are shown in Figure
1
b. Each conjugation reaction proceeded in good yield and gave products of
acceptable purity (>90%) based on HPLC and ESI-MS analyses.
Because of the relatively low binding affinities of these modified MPOs (see
Materials and Methods) we decided to conduct our cleavage experiments at 25oC. The complementary RNA target strand (Fig.
1
b) was synthesized and labeled at the 5'-terminus with
32
P using [[gamma]-
32
P]ATP and T4 polynucleotide kinase. An excess of modified MPO was employed in
each annealing reaction to drive the equilibrium to the duplex form at this
temperature. Our first set of experiments examined the ability of G-1719 to direct site-specific cleavage on the RNA strand in the presence of an exogenous
catalyst (1 M EDA, pH 7-8). As shown in Figure
2
(lanes 1 and 2), incubation of single-stranded RNA with the EDA catalyst produces a random cleavage pattern,
whereas a single site-specific cleavage is observed in the presence of G-1719. Control experiments using a fully complementary MPO (i.e. not
containing the non-nucleotide linker) showed complete protection of the RNA strand against
hydrolytic cleavage (M.A.Reynolds and T.A.Beck, unpublished results). By
correlating the bands in lane 1 with the known sequence for the RNA target it
is evident that the primary cleavage fragment seen in lane 2 occurs in the
vicinity of the non-nucleotide linker. Furthermore, the intensity of this band is greater than
the corresponding band in lane 1, indicating that this site is cleaved at a
faster rate when oligonucleotide G-1719 is present. Thus the heteroduplex formed between the RNA target and G-1719 creates a site on the RNA strand that is rendered more
sensitive to catalytic hydrolysis compared with the single-stranded control. Also, according to our data the base paired regions of
the RNA strand are protected from hydrolytic cleavage, confirming the
hypothesis that base stacking slows down the rate of backbone hydrolysis.
A number of investigators have reported linking synthetic RNA cleaver catalysts
to oligonucleotides (
31
-
38
). In most cases these catalysts were tethered to the 5'- or 3'-terminus. Here we directed the catalyst to a site on
the RNA that is rendered more sensitive to hydrolysis due to the replacement of
one of the complementary bases with a non-nucleotide-based linker. Our expectation was that the ribonucleotide base
opposite this linker would be oriented outward from a stacked helical geometry,
thereby rendering its phosphodiester bonds more susceptible to acid/base
hydrolysis.
Other investigators have reported differences in the rate of acid/base
hydrolysis of single- versus double-stranded RNA. For example, an imidazole-based catalyst was shown to cleave tRNA preferentially at
junctions between stem and loop regions; double stranded regions are relatively
unaffected (
15
). Such results are consistent with the prediction that double-stranded RNA is resistant to hydrolysis due to the restricted orientation
of the 2'-hydroxyl of the first base with respect to the 5'-bonded oxygen of the second base (
17
). This is also consistent with an in-line displacement mechanism proposed for the cleavage reaction catalyzed
by RNase A (
39
).
According to our data an MPO containing a non-nucleotide-based linker in place of one of the nucleotide bases directs a site-specific cleavage reaction on its complementary RNA target in
the presence of exogenous catalyst (in this case 1 M EDA). Furthermore,
modified MPOs G-2435 and G-2437 promote site-specific cleavage in the absence of exogenous catalyst. Thus
we have demonstrated that the covalent attachment of a catalyst to an
oligonucleotide via a non-nucleotide-based linker enables it to be directed toward a hydrolytically
sensitive site on the RNA target, whereupon site-specific cleavage occurs. The rates of these cleavage reactions are
relatively modest however (
t
½
values are estimated to be >5 days at 25oC based on inspection of the autoradiographs). This is believed to be due
in part to the low binding affinity of these modified MPOs. Other work in our
laboratory has demonstrated that backbone-modified oligonucleotides containing alternating
Rp
chiral methylphosphonate/phosphodiester linkages have significantly higher
binding affinities for their RNA targets (unpublished results). We anticipate
that similar conjugates including this improved backbone will yield more
acceptable cleavage rates at physiological temperatures. Other catalysts are
also being investigated.
We thank Richard Hogrefe and Lina Borozdina for providing the synthetic RNA
target and some of the methylphosphonate oligonucleotides. We thank John Jaeger
for critically reviewing this manuscript and also thank Jik Chin for helpful
scientific discussions. We are grateful to Timothy Riley and Michelle Chapman-Scurria at JBL Scientific Inc. for providing some of the methylphosphonate
oligonucleotides and for running the ESI-MS analyses respectively.
REFERENCES
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