Enhancing Sequence-Specific Cleavage of RNA within a Duplex

Enhancing Sequence-Specific Cleavage of RNA within a Duplex Region: Incorporation of 1,3-Propanediol Linkers into Oligonucleotide Conjugates of Serino...
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Bioconjugate Chem. 2001, 12, 900−905

Enhancing Sequence-Specific Cleavage of RNA within a Duplex Region: Incorporation of 1,3-Propanediol Linkers into Oligonucleotide Conjugates of Serinol-Terpyridine Bobby N. Trawick,† Todd A. Osiek,† and James K. Bashkin*,‡ Department of Chemistry, Washington University, Campus Box 1134, St. Louis, Missouri 63130-4899. Received February 15, 2001; Revised Manuscript Received May 25, 2001

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)serinol-terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic-RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5′- and 3′-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol-terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA-DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.

INTRODUCTION

As the impact of scientific research at the interface of chemistry and biology continues to expand, so does our recognition of the potential that exists in the area of biological mimicry by synthetic reagents. One such area of interest today is the development of synthetic mimics of ribozymes (or ribozyme mimics). Since the earliest reports of sequence-specific RNA cleavage by this unique class of bioconjugates (1-5), much effort has been devoted to the establishment of structure-activity relationships for these reagents (2, 6-15). One of the principal goals is to achieve catalytic cleavage of specific RNA substrates with efficiencies approaching and/or exceeding those of natural ribozymes and ribonucleases (16). A ribozyme mimic is typically synthesized by the covalent attachment of an RNA cleavage catalyst to a synthetic DNA strand (or DNA analog) via a linker arm. The catalyst is usually a metal complex or nitrogen-containing organic molecule that catalyzes the scission of RNA phosphodiester bonds via transesterification and/or hydrolysis reactions. The DNA probe serves as the recognition domain of the ribozyme mimic, and the catalyst serves as the active site. Through the convenience of automated DNA synthesis and the specificity of complementary Watson-Crick base pairing, the DNA strand can be custom-tailored to direct the RNA cleaving capability of the catalyst toward any RNA target of known base sequence. The central concept behind ribozyme mimic development is that upon selectively binding to its complementary RNA sequence, the mimic will deliver the attached RNA cleavage catalyst across the major or minor groove * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: MetaPhore Pharmaceuticals, Inc., 1910 Innerbelt Business Center Drive, St. Louis, MO 63114. ‡ Current address: Pharmacia Corporation, Mail Zone R3A, 800 N. Lindbergh Blvd., St. Louis, MO 63167.

of the DNA-RNA duplex and within sufficient proximity of the RNA phosphodiester backbone to effect the RNA transesterification reaction. Theoretically, the ribozyme mimic should disengage from the resultant RNA product fragments and subsequently bind to and cleave another complementary RNA target sequence. This would in effect create a catalytic cycle of specific RNA destruction. Our approach over the last decade (1, 13, 16, 17), coupled with Magda’s recently proven strategy (6) for achieving catalytic activity with ribozyme mimics, encourages placement of the catalyst at an internal position within the ribozyme mimic DNA strand rather than simply tethering the catalyst to the end of the sequence. This is because cleavage of the RNA target within the duplex region is more favorable for product release, given that cleavage outside of the duplex region will be plagued by product inhibition. One of the major concerns with this particular strategy, however, is the ability of ribozyme mimics to cleave the RNA residues within the doublestranded regions of DNA-RNA hybrids (18). RNA in a DNA-RNA duplex is relatively inert to cleavage by metal ions and complexes in comparison to its single-stranded form (19). This is likely a consequence of the conformational rigidity that is characteristic of duplexed RNA. In fact, this pronounced difference in cleavage susceptibility between single- and double-stranded RNA has been exemplified by pioneering efforts from several research groups. For example, Kolasa et al. demonstrated that when a short DNA oligonucleotide was annealed to a segment of t-RNAPhe, the complementary RNA sequence was protected from cleavage by Eu3+- and La3+-based RNA cleavage catalysts (18). The results of this study led the authors to conclude that in the design of ribozyme mimics, it may be more advantageous to place the catalyst at the end of the DNA strand (proximate to single-stranded RNA) rather than at an internal position where the RNA is duplexed by DNA and inert to cleavage. In a related study, Hegg et al. investigated the

10.1021/bc0100197 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/12/2001

Enhanced RNA Cleavage in a Duplex Region

cleavage of RNA hairpin structures by a macrocyclic Cu2+ complex (20). Cleavage occurred in both the single- and double-stranded regions of the RNA hairpins; however, the amount of cleavage in double-stranded regions was not as pronounced as that observed in single-stranded regions. In another relevant study, Huesken et al. showed that RNA bulges in DNA-RNA duplexes are highly susceptible to cleavage by Mg2+ ion and La3+ hexadentate-Schiff base macrocycles (21). The authors concluded that the single-stranded RNA in the bulged region has a high degree of conformational freedom, which allows the RNA to adopt the in-line conformation required for the transesterification reaction. On the basis of these findings, the same research group designed ribozyme mimics that would place lanthanide-based RNA cleavage catalysts directly opposite induced bulges in the RNA target strand (11). These mimics were found to be 13 times more reactive than those that formed perfect duplexes with the RNA target. Most recently, this group reported ribozyme mimics that function with multiple turnover (12). In that study, lanthanide metal complexes were covalently linked to the 5′-ends of 2′-methoxyethyl (MOE) oligonucleotides. The key to this approach was that the oligonucleotide portions of the ribozyme mimics were designed such that upon binding to the RNA target, a bulge would be formed in the RNA strand near the center of the DNA-RNA duplex region. The attached catalyst could subsequently be folded back to preferentially cleave the single-stranded RNA in the bulged region. This creative design yielded catalytic turnover with the 2′-MOE conjugates, but no evidence of catalytic activity was observed with conjugates comprised of unmodified DNA probes. In an analogous context, we demonstrated that the extent of sequence-specific RNA cleavage by Cu(II) terpyridine-based ribozyme mimics could be substantially increased using the concept of minimal nucleotide replacement (13). In that study, serinol-terpyridine modification 2 was incorporated in place of a single nucleotide residue in the ribozyme mimic DNA strand. The serinolterpy effectively served as an artificial abasic site, which (upon DNA-RNA duplex formation) left an unpaired RNA residue directly opposite the site of the modification. The increased level of conformational freedom provided by the serinol-terpyridine modification rendered its opposing RNA residue much more susceptible to cleavage than when in the fully base-paired state and resulted in a greater than 3-fold increase in overall RNA cleavage efficiency in comparison to our first-generation, fully duplexed ribozyme mimics based on modification 1. The work described in this report represents the next logical step in our group’s efforts to design ribozyme mimics that exploit the enhanced cleavage susceptibility of single-stranded RNA over double-stranded RNA. We have designed a new series of ribozyme mimics that yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst (Cu(II)-terpy) upon duplex formation. This design effect was achieved using the 1,3propanediol linker 3, which, like serinol, mimics the three-carbon spacing between the 5′- and 3′-hydroxyls of a natural nucleotide. Incorporation of single and multiple linkers at positions adjacent to a serinol-terpyridine modification in the DNA strand resulted in perturbations to the opposing RNA backbone that allowed for the cleavage of multiple phosphodiester linkages by metalcatalyzed transesterification and/or hydrolysis reactions. The most active ribozyme mimic in the series exhibited cleavage of multiple equivalents of its complementary

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RNA substrate, revealing a catalytic rather than stoichiometric mode of action for this reagent. EXPERIMENTAL PROCEDURES

Synthesis of Ribozyme Mimics. The parent DNA sequence and the conjugates containing the serinolterpyridine or 1,3-propanediol linker modifications (Figure 2) were prepared by automated DNA synthesis on an ABI Model 380B DNA synthesizer. The modified phosphoramidite building blocks 2 and 3 were placed on two of the free auxiliary ports of the synthesizer and incorporated at appropriate positions in the synthesis protocols. The detailed synthesis of phosphoramidite 2 has been previously reported by our group (13). Linker phosphoramidite 3 was purchased from Glen Research and used as received. All oligonucleotides were purified by UV-shadow excision from preparative 20% denaturing polyacrylamide gels, followed by elution from the gel fragment and ethanol precipitation. The structures of probes 1-7 were confirmed by negative-ion MALDI-TOF mass spectrometry (Table 1). The concentrations of the oligonucleotide conjugates were determined by UV absorbance at 260 nm utilizing molar extinction coefficients () calculated with the Oligo Analyzer program (version 2.0) from Integrated DNA Technologies (IDT). [ (L/mol‚ cm): probe 1, 283,700; probe 2, 276,500; probe 3, 269,000; probe 4, 276,500; probe 5, 266,600; probe 6, 269,300; probe 7, 252,500]. Preparation of 3′-End-Labeled 31-mer RNA Target Sequence. The synthetic 31-mer RNA sequence (5′AAACCAACCCUUCAGAGACUCUGUCGACGGG-3′) was purchased from IDT. The RNA was 3′-end labeled using R-32P-dATP (Amersham) and poly(A) polymerase (USB) according to standard procedures (22). Note that this procedure adds a single dA residue to the 3′ end of the RNA, increasing the total length to 32 nucleotides. The labeled RNA was phenol extracted, ethanol precipitated, and purified by excision from a 20% denaturing polyacrylamide gel. The RNA was then eluted from the gel fragment, ethanol precipitated, and diluted to the appropriate concentrations with RNase-free water. RNA concentrations were determined by UV absorbance at 260 nm using the molar extinction coefficient calculated using the Oligo Analyzer program (version 2.0) from IDT [ (L/ mol‚cm): 302 500].

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Table 1. MALDI-TOF Mass Spectral Analysis of Conjugates probe

calcd mass [M - H]-1

obsvd mass [M - H]-1

1 2 3 4 5 6 7

9719 9568 9402 9568 9402 9417 9084

9728 9556 9427 9562 9397 9407 9078

Sequence-Specific RNA Cleavage Reactions. RNA cleavage reactions were carried out in a total volume of 10 µL containing 10 mM (N-[2-hydroxyethyl]piperazineN′-[2-ethanesulfonic acid]) (HEPES) buffer (pH 7.4), 0.1 M NaClO4 (to control ionic strength), 250 nM 31-mer RNA, 5 µM ribozyme mimic, and 5 µM CuSO4. The ribozyme mimic and CuSO4 were premixed and added to a solution of the RNA, buffer, and NaClO4. The reactions tubes were tightly sealed and completely submerged in a 45 °C water bath for 40 h. Quenching of the reactions was achieved through the addition of 10 µL gelloading buffer. Aliquots of 10 µL were run on 20% denaturing polyacrylamide sequencing gels, alongside partial RNase T1 digests and base hydrolysis lanes for mapping of the RNA cleavage sites. The electrophoresis images were quantified using a Molecular Dynamics PhosphorImager and the ImageQuant software package (Version 5.0). Catalytic Cleavage of RNA with Probe 6. The sequence-specific RNA cleavage reactions with probe 6 in the presence of excess RNA target were carried out in a total volume of 10 µL containing 10 mM (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (HEPES) buffer (pH 7.4), 0.1 M NaClO4, 125 nM probe 6, 125 nM CuSO4, and either 250 nM, 500 nM, 1.0 µM, or 1.25 µM of 31-mer RNA. The ribozyme mimic and CuSO4 were premixed and added to a solution containing the RNA, buffer, and NaClO4. The reactions tubes were tightly sealed and completely submerged in a water bath at 45 °C for 40 h, after which the reactions were quenched by the addition of 10 µL gel-loading buffer. Aliquots of 10 µL were run on 20% denaturing polyacrylamide sequencing gels, and the electrophoresis images were quantified using a Molecular Dynamics PhosphorImager and the ImageQuant software package (Version 5.0). RESULTS AND DISCUSSION

The structures of the ribozyme mimics employed in this study are detailed in Figure 1. Figure 2 is a representative autoradiograph of a 20% denaturing polyacrylamide gel obtained after treatment of the 3′-32P-end labeled 31mer RNA target with probes 1-7. In these experiments, ribozyme mimics containing from zero to four linkers surrounding the serinolterpyridine modification were tested for sequence-specific RNA cleavage activity in the presence of Cu2+ ion. For each probe, the serinol-terpy modification was located at position 16 (from the 5′-end) of the 31-mer DNA sequence. The 1,3-propanediol linkers were placed to the 3′-side, to the 5′-side, and to both sides of the modification simultaneously. The ribozyme mimic and RNA target were incubated in the presence of Cu2+ for 40 h at 45 °C and pH 7.4. For electrophoretic analyses, RNA cleavage lanes were run alongside partial RNase T1 digests and base hydrolysis lanes to allow mapping of the exact sites of cleavage produced by the different mimics. The relative RNA cleavage percentages of the probes are denoted in Figure 2.

The ribozyme mimics employed in this study have structural differences in their respective DNA strands that yield unique RNA cleavage patterns for each reagent (Figure 1). The patterns are clearly governed by the locations of the 1,3-propanediol linkers in the DNA sequences. Probe 1, which contains a single serinol terpyridine modification in the middle of the DNA strand (16th position), cleaves the RNA phosphodiester linkages directly opposite the expected location of the catalyst upon RNA-DNA duplex formation. The serinol-terpy modification of the DNA strand is located directly opposite nucleotide rA16 in the complementary RNA strand, and the major site of cleavage is observed at this position as expected. There is also a minor cleavage band at residue rG15 to the 5′ side of the cleavage site. These results were consistent with our previous observations using these types of reagents (13). Cleavage of the RNA target with probe 2, which contains a single three-carbon linker to the 3′-side of the serinol-terpy in the DNA strand (directly across from rG15), yielded slightly different results. The major site of cleavage remained at nucleotide rA16; however, the number of minor cleavage bands increased toward the 5′-side of the RNA. A minor cleavage band was observed at nucleotide rA14 in addition to the minor band at rG15. There was also a slight increase in the amount of cleavage at position rG15 in comparison to probe 1. Incorporation of two linkers to the 3′-side of the serinol-terpy residue in the DNA strand (probe 3) results in an even further extension of the number of minor cleavage bands to the 5′-side of the major cleavage site (rA16) in the RNA strand. In this case, the amount of cleavage at rA14 increased, but the amount of cleavage at rG15 did not (in comparison to probe 2). For comparison, we investigated the effects of placing the linker to the 5′-side of the serinol-terpy modification in the DNA strand. Cleavage of the RNA target with probes 4 and 5, which contain one and two linker(s) to the 5′-side of the serinol-terpy respectively, yielded results similar to those obtained with the analogous probes 2 and 3. Nucleotide rA16 was the major site of cleavage, and rG15 was a minor site of cleavage in each case. probe 4, which was designed to place a linker across from nucleotide rG17 in the RNA strand, led to an additional minor cleavage band at position rG17. Probe 5, which was designed to place linkers directly opposite positions rG17 and rA18 yielded two additional minor cleavage bands at these locations as expected. To provide further corroboration of the utility of this approach, we prepared ribozyme mimics having linkers on both the 3′- and 5′- sides of the serinol-terpy modification in the DNA strand. Probe 6 consisted of one linker to the 3′-side of the modification, in addition to one linker to the 5′-side of the modification. Interestingly, cleavage of the RNA target with this reagent revealed a shift in the major site of cleavage from nucleotide rA16 (as observed with probes 1-5) to nucleotide rG15- a onenucleotide shift toward the 5′-end of the RNA sequence. Minor cleavage bands were also observed at nucleotides rA14, rA16, rG17, and rA18. These cleavage products essentially represented a combination of the cleavage products obtained separately with probes 2 and 4, except that the major site of RNA cleavage was shifted by one nucleotide. A similar phenomenon was observed with probe 7, which contains two linkers to the 3′-side of the serinol-terpy as well as two linkers to the 5′-side of the serinol-terpy. In this case, the major site of cleavage was shifted even further to the 5′-side of the RNA to position rA14. Minor cleavage bands were also observed at nucle-

Enhanced RNA Cleavage in a Duplex Region

Bioconjugate Chem., Vol. 12, No. 6, 2001 903

Figure 1. Structures of the ribozyme mimics and 31-mer RNA target sequence employed in this study. The “X” indicates the location of the serinol-terpyridine modification in the DNA strand, while the “S” represents the 1,3-propanediol linker modification. The 31-mer RNA target sequence is shown in bold text.

otides rC13, rG15, rA16, rG17, and rA18, which represents a combination of the cleavage products obtained with probes 3 and 5 separately. In this instance, however, the major site of cleavage was shifted by two nucleotides toward the 5′-end of the RNA target. The observed shifts in the major site of cleavage with probes 6 and 7 may offer some structural insights into the orientation of the serinol-terpyridine modification in the DNA-RNA duplex. When the degree of flexibility is equal on both sides of the serinol-terpy modification, cleavage occurs preferentially toward the 5′-side of the RNA strand, suggesting a preference for delivery of the catalyst across the larger major groove of the DNA-RNA duplex. In addition to the sequence-specific RNA cleavage reactions using modified probes as described above, control experiments were conducted with the parent and control probes (Figure 1). Both of these probes lack the serinol-terpyridine modification in their DNA strands and hence possess no RNA cleavage capabilities. The parent probe is simply the full complement of the 31mer RNA target sequence employed in this study. As

expected, no cleavage of the RNA target was effected by this probe in the presence of Cu(II) under our standard assay conditions (Figure 2, lane 4). The control probe, which contains four consecutive 1,3-propanediol linkers in the center of the DNA strand, also effected no significant cleavage of the RNA target (