Bioconjugate Chem. 2004, 15, 1275−1280
1275
Sequence-Selective Cleavage of Oligoribonucleotides by 3d Transition Metal Complexes of 1,5,9-Triazacyclododecane-Functionalized 2′-O-Methyl Oligoribonucleotides Teija Niittyma¨ki and Harri Lo¨nnberg* Department of Chemistry, University of Turku, FIN-20014 Turku, Finland. Received July 14, 2004; Revised Manuscript Received September 10, 2004
2′-O-Methyl oligoribonucleotides bearing a 3′-[2,6-dioxo-3,7-diaza-10-(1,5,9-triazacyclododec-3-yl)decyl phospate conjugate group have been shown to cleave in slight excess of Zn2+ ions complementary oligoribonucleotides at the 5′-side of the last base-paired nucleotide. The cleavage obeys first-order kinetics and exhibits turnover. The acceleration compared to the monomeric Zn2+ 1,5,9-triazacyclododecane chelate is more than 100-fold. In addition, 2′-O-methyl oligoribonucleotides having the 1,5,9-triazacyclododec-3-yl group tethered to the anomeric carbon of an intrachain 2-deoxy-β-D-erythropentofuranosyl group via a 2-oxo-3-azahexyl, 2,6-dioxo-3,7-diazadecyl, or 2,9-dioxo-3,10-diazatridecyl linker have been studied as cleaving agents. These cleave as zinc chelates a tri- and pentaadenyl bulge opposite to the conjugate group approximately 50 times as fast as the monomeric chelate and show turnover. The cleavage rate is rather insensitive to the length of linker. Interestingly, a triuridyl bulge remains virtually intact in striking contrast to a triadenyl bulge. Evidently binding of the zinc chelate to a uracil base prevents its catalytic action. Replacement of Zn2+ with Cu2+ or Ni2+ retards the cleaving activity of all the cleaving agents tested.
INTRODUCTION
Oligonucleotide conjugates that sequence-selectively cleave complementary RNA targets have attracted considerable attention during the past decade (1-5). Such cleaving agents may enable chemical tailoring of RNA and, hence, serve as artificial restriction enzymes for RNA in vitro. In addition, they may possibly find applications in chemotherapy as chemically reactive antisense oligonucleotides that are able to destroy their target mRNA and exhibit RNase H independent turnover. The feasibility of such of an approach has been demonstrated by in vitro studies on cell lines (6, 7), although not in vivo. Most of the artificial ribonucleases described are conjugates of metal ion chelates having a lanthanide ion (8-15), Cu2+ (16-19), or Zn2+ (20-22) as the central ion. In addition, conjugates of peptidelike oligomers have been reported to catalyze sequence-selective phosphodiester hydrolysis, but their catalytic activity is still low compared to that of the metal ion-dependent cleavers (23-31). Since certain macrocyclic polyamines, the socalled azacrowns, bind 3d transition metal ions very tightly (32-33), in fact more tightly than many of the previously described metal-ion-dependent cleaving agents, and since some of these chelates, above all 1,5,9-tirazacyclododecane ([12]aneN3, 1), have been shown to promote the hydrolysis of RNA fragments rather efficiently (34-37), azacrown-functionalized oligonucleotides may be regarded as viable candidates of artificial RNases. For this purpose, we have recently prepared 2′-O-methyl oligoribonucleotides bearing a 1,5,9-triazacyclododec-3yl group tethered either to the 3′-terminus of the oligo* To whom correspondence should be addressed. E-mail:
[email protected].
nucleotide or to an intrachain abasic sugar unit (38). We now report on evaluation of the applicability of their Zn2+ complexes as artificial RNases. A detailed kinetic analysis of the cleavage reaction has been carried out by using synthetic oligoribonucleotides as targets. The influences of the concentration of the target and cleaving agent, the structure of the linker connecting the azacrown to the oligonucleotide, and the size and base content of the bulge created on the target upon hybridization have been studied. In addition, some comparative measurements with Cu2+ and Ni2+ complexes have been carried out. EXPERIMENTAL PROCEDURES
Materials. Preparation of the azacrown-functionalized 2′-O-methyl oligoribonucleotides 2-4 and 6 has been described previously (38). Conjugate 5 was obtained as reported for 6, and its authenticity was verified by ESI-MS. MS(ESI) for 5: m/z 587.6 (8%) [M - 8H]8-, 671.9 (30%) [M - 7H]7-, 783.8 (76%) [M - 6H]6-, 940.7 (100%) [M - 5H]5-, 1175.9 (94%) [M - 4H]4-, 1568.0 (44%) [M - 3H]3-. Mobs 4708.5, Mcalcd 4707.4. The chimeric 2′-O-methylribo/ribo oligonucleotides (7-17) used as targets were assembled from commercially available 2′-O-methyl and 2′-O-triisopropylsilyloxymethyl (2′-O-TOM) protected 2-cyanoethyl-N,Ndiisopropylphosphoramidite building blocks (Glen Research) by conventional phosphoramidite strategy using a 1.0 µmol scale and following the standard RNA-coupling protocol of Applied Biosystems 392 DNA/RNA Synthesizer. The buffer reagents and salts employed were of reagent grade. All buffer solutions were prepared in sterilized water, and sterilized equipments were used for their handling. Kinetic Measurements. The reactions were carried out in Eppendorf tubes immersed in a water bath, the
10.1021/bc0498323 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/02/2004
1276 Bioconjugate Chem., Vol. 15, No. 6, 2004
Niittyma¨ki and Lo¨nnberg
Chart 1. Structures of Cleaving Agents (1-6), Targets (7-11), and Potential Product Oligonucleotides (12-17)a
a
Bold letters refer to ribonucleotides, the rest to 2′-O-methylribonucleotides.
temperature of which was maintained at 35.0 ( 0.1 °C. The volume of the reaction mixture was 200 µL. The pH was adjusted to 7.3 with a HEPES buffer (0.1 mol L-1). The Zn2+, Cu2+ and Ni2+ ions were added as nitrates to give a total metal ion concentration of 50 µmol L-1, and the ionic strength was adjusted to 0.1 mol L-1 with sodium nitrate. The concentration of the azacrownfunctionalized oligonucleotides and their targets was varied from 9 to 36 µmol L-1 and 18 to 36 µmol L-1, respectively. p-Nitrobenzenesulfonate ion was used as an internal standard. Aliquots of 20 µL were withdrawn at suitable intervals and immediately cooled to 0 °C. The reaction was quenched by adding aqueous hydrogen chloride (1.0 µL of 1.0 mol L-1 solution). The samples were analyzed immediately. Analysis of Samples. The samples were analyzed by capillary zone electrophoresis (CZE; Beckman Coulter P/ACE MDQ CE System) using a fused silica capillary (50 µm inner diameter, 60.2 cm total length, 50 cm effective length). The inverted polarity and citrate buffer (0.2 mol L-1, pH 3.1) were used. The voltage applied was -30 kV. The temperature of the capillary was kept at 25 °C. The samples were injected using hydrodynamic injection with 2 psi for 8 s. The capillary was flushed with water, 10 mmol L-1 aqueous hydrogen chloride, and the background electrolyte buffer (for 3 min each) between every analytical run. UV-detection at 254 nm was applied to quantify the oligonucleotides and the internal standard. Calculation of Rate Constants. First-order rate constants for the cleavage of the target oligonucleotides
were calculated by applying the integrated first-order rate law to the disappearance of the starting material. The peak area was first normalized by dividing the area by the migration time and then by the similarly normalized area of the internal standard. Determination of the Cleavage Site. The cleavage site within target 10 was determined by spiking with standards 12-17. Each of the standards was added one after another to an aliquot of the reaction mixture, which contained the cleaved target 10, and the mixtures were analyzed by CZE as described above. Standards 13, 14, and 17 did not comigrate with the cleavage products. Standard 16 comigrated with the minor product of the cleavage reaction. Standards 12 and 15 both comigrated with the major product. For this reason, those mixtures were additionally analyzed by an otherwise similar CZE method but using a different background electrolyte, viz. iminodiacetic acid (50 mmol L-1, pH 2.3). Measurements in this more acidic buffer revealed standard 15 to be identical with the major cleavage product. RESULTS AND DISCUSSION
Structure of the Cleaving Agents and Their Target Oligonucleotides. Chart 1 shows the cleaving agents studied (2-6), their target oligonucleotides (7-11), and the oligonucleotide standards used for the determination of the cleavage site (12-17). The cleaving agents are fully 2′-O-methylated oligoribonucleotides bearing the catalytically active [12]aneN3 chelate tethered either to an abasic sugar unit within the chain (2-4) or to the 3′-terminal hydroxy function (5, 6). The
Sequence-Selective Cleavage of Oligoribonucleotides
Bioconjugate Chem., Vol. 15, No. 6, 2004 1277
Figure 1. Cleavage of chimeric 2′-O-methylribo/ribo oligonucleotide 10 by sequence-selective cleaving agent 5 in 0.1 mol L-1 HEPES buffer at pH 7.3 and 35 °C (I ) 0.1 mol L-1 with NaNO3), and the electropherograms obtained at time 0, 165, and 530 h. The initial concentration of both the target oligonucleotide and the cleaving agent is 36 µmol L-1.
targets are chimeric 2′-O-methylribo/ribo oligonucleotides containing several unsubstituted ribo units in the vicinity of the expected cleavage site, i.e. either 6 or 8 ribonucleosides in the middle of an 2′-O-methylribonucleotide chain (7-9) or 11 ribonucleosides at the 5′-end of a 2′-Omethylribonucleotide chain (10, 11). The base sequences are selected so that upon hybridization of the cleaving agent with target, either a tri- or pentanucleotide bulge is formed on the target sequence (binding of 2-4 to 7-9) or the sequence of the cleaving agent is fully complementary with the 3′-terminal sequence of the target (binding of 5 to 10 or 6 to 11). Creation of a bulge to achieve intrachain cleavage is essential, since it has been shown that [12]aneN3 cleaves oligoribonucleotide within a bulged region, even at a single nucleotide bulge, but it is unable to cleave phosphodiester bonds within a double helical region (36, 37). Insertion of 2′-O-methylribonucleosides in the target sequence facilitates the synthesis and ensures high affinity of the target to the cleaving agent, preserving the A-type duplex structure (39). Cleavage by the 3′-Tethered Conjugates. Figure 1 shows as an illustrative example the cleavage of target 10 by conjugate 5 in 0.1 mol L-1 HEPES buffer at pH 7.3 and 35.0 ( 0.1 °C. The concentration of both oligomers (5,10) was 36 µmol L-1 and that of Zn2+ somewhat higher, about 50 µmol L-1, to ensure complete complexing [the stability constant for Zn2+[12]aneN3 is 108.6 L mol-1 (33) ]. Increasing of the Zn2+ concentration to 150 µmol L-1 did not, however, have any influence on the cleavage rate. The ionic strength was adjusted to 0.1 mol L-1 with NaNO3. The progress of the reaction was followed by
determining the composition of the aliquots withdrawn at suitable intervals by capillary electrophoresis. To facilitate the quantification, p-nitrobenzenesulfonate was used as an internal standard. As seen from the electropherograms in Figure 1, the target 10 entirely disappeared and a product oligonucleotide appeared at a longer retention time. First-order kinetics were reasonably well obeyed. The cleavage site within target 10 was determined by spiking the product mixture with the potential cleavage products, i.e. with separately synthesized chimeric 2′-Omethylribo/ribo oligonucleotide standards 12-17. Each of the standards was mixed separately with an aliquot of the reaction mixture containing the cleaved target 10. The major product of the cleavage reaction, representing about 80% of the total cleavage, was observed to be 15, the minor product being 16. Accordingly, target 10 is predominantly cleaved at the 5′-side of the last basepaired nucleotide and to a minor extent at the phosphodiester bond one nucleotide toward the 5′-end. The cleavage reaction also shows turnover. As seen from Figure 2, the target oligonucleotide is entirely cleaved obeying first-order kinetics, even when the target is present in a 4-fold excess compared to the cleaving agent. Cleaving agent 6 that contains only three cytosine bases and one guanine base and, hence, binds to its target (11) less firmly than 5 that forms eight CG base-pairs with 10, exhibits under these turnover conditions 5-fold cleaving activity compared to 5. Most likely the enhanced cleavage does not, however, result from the reduced affinity to the target, but from the fact that an 5′-UpA-3′ bond is cleaved instead of an 5′-ApG-3′. It is known that
1278 Bioconjugate Chem., Vol. 15, No. 6, 2004
Niittyma¨ki and Lo¨nnberg Table 2. First-Order Rate Constants for the Cleavage of Targets 7-9 by Zn2+ Chelates of Conjugates 2-4 in 0.10 Mol L-1 HEPES Buffer at pH 7.3 and 35 °C (I ) 0.1 Mol L-1 with NaNO3)a
Figure 2. Cleavage of chimeric 2′-O-methylribo/ribo oligonucleotide 10 by cleaving agent 5 (squares) and 11 by 6 (circles). The concentration of the target oligonucleotides (10,11) is 36 µmol L-1 and that of the cleaving agents (5,6) 9 µmol L-1. For the conditions, see the legend of Figure 1. Table 1. First-order Rate Constants for the Cleavage of Targets 10 and 11 by Metal Ion Chelates of Conjugates 5 and 6, Respectively, in 0.10 Mol L-1 HEPES Buffer at pH 7.3 and 35 °C (I ) 0.1 Mol L-1 with NaNO3)a cleaving agent
target
M2+
5
10
Zn2+
5 5 6
10 10 11
Cu2+ Ni2+ Zn2+
6 6
11 11
Cu2+ Ni2+
c (cleaving agent), µmol L-1
c (target), µmol L-1
36 18 18 9 18 18 18 9 9 9
36 18 36 36 18 18 36 36 36 36
k,
10-6
s-1
cleaving agent
target
2 2 2 3
7 8 9 7
3
8
3 4
9 7
4
8
4
9
within many sequences, the inherent hydrolytic stability of the 5′-UpA-3′ bond is lower than that of the other phosphodiester bonds (40). Table 1 records the first-order rate constants obtained for the cleavage of targets 10 and 11 with conjugates 5 and 6, respectively. With the more stable pair of the cleaving agent and target, 5/10, the reaction rate is increased with the increasing concentration of 5. Although the reaction at each concentration of 5 shows turnover, the rate is not strictly proportional to the concentration of 5, but the rate constants obtained in excess of the target 10 are only half of the values expected on the basis of first-order dependence of the rate on the concentration of 5. The reaction possibly is subject to moderate product inhibition, suggesting that the cleaving agent hybridizes slightly more efficiently with the product oligonucleotides 15, 16 than with the target 10. This inhibition is not, however, sufficiently strong to markedly deviate the cleavage from first-order kinetics. At 1:1 concentration ratio of the cleaving agent 5 and target 10, the cleavage rate is independent of the concentration, as shown by the fact that the rate constants obtained at 36 and 18 µmol L-1 are equal within the limits of experimental error. Evidently in this concentration range the duplex formation is virtually quantitative. Surprisingly, the less efficiently hybridizing cleaving agent/target pair 6/11 behaved quite differently. Increasing the concentra-
c (target), µmol L-1
k, 10-6 s-1
18 18 9 18 9 18 9 9 18 9 18 9 9
18 18 36 18 36 18 36 36 18 36 18 36 36
0.74 ( 0.06 1.2 ( 0.08
