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Nucleic Acids Research, 2001, Vol. 29, No. 10 2199-2204
© 2001 Oxford University Press

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts

William C. Putnam, Andrew T. Daniher, Bobby N. Trawick and James K. Bashkin*

Department of Chemistry, Washington University, St Louis, MO 63130-4899, USA

Received October 12, 2000; Revised January 29, 2001; Accepted March 10, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phananthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The development of artificial nucleases (1–8) is an area of great interest in chemistry and biology today. Ribozyme mimics (see below) form one class of such biomimetic reagents (9–14). They employ a DNA (or DNA analog) strand for molecular recognition and an RNA cleavage agent for activity (typically a metal complex that catalyzes the transesterification and/or hydrolysis of RNA). Therefore, these molecules are functional mimics of ribozymes in which the large catalytic domain of the natural ribozyme has been replaced by a small molecule catalyst, and where the requirement for a properly folded tertiary structure has been eliminated. As such, ribozyme mimics effectively decouple binding and catalytic domains, and allow fundamental investigations of these separate functions.

The first example of a wholly synthetic ribozyme mimic was reported by our group (15) (Fig. 1A), with nearly simultaneous important contributions from other groups in the area (16–19). There has been continued cross-fertilization of this field as important discoveries have been reported by academic and industrial groups (9,20–24). We have been particularly concerned with developing general principles that govern cleavage efficiency, and how and where cleavage is directed by specific molecular designs (i.e. attack via the major or minor grooves; Fig. 1) (25).



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  		Figure 1. Examples of the catalytic cores of several ribozyme mimics. (A) First example of a wholly synthetic ribozyme mimic, a major-groove-directed terpy conjugate. (B) A major-groove-directed bipyridine conjugate. (C) A minor-groove-directed bipyridine conjugate. (D) A minor-groove-directed terpy conjugate.

 
Our initial ribozyme mimic was constructed by the covalent incorporation of Cu(II)–terpyridine [Cu-terpy, a well-known (26–32) RNA transesterification/hydrolysis agent] into a DNA 17mer via attachment at the C-5 position of 2'-deoxyuridine (15). The cleavage efficiency of Cu-terpy-based ribozyme mimics was later improved by attaching terpy to serinol, a ‘minimal nucleotide replacement’ (24,33,34). Serinol removes one Watson–Crick base pair and increases local flexibility at the site of cleavage, which may enhance the formation of the phosphorane geometries required for nucleophilic cleavage of phosphodiesters.

Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phen- and terpy-derived reagents. These results were demonstrated with a 28mer RNA target, which allowed ready identification of the cleavage products as true transesterification products, and with a 159mer fragment of the HIV gag gene mRNA, which demonstrated the precise specificity of cleavage in the presence of many competing sites.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Compound 2 (serinol–neocuproine phosphoramidite building block)
Neocuproine was nitrated (8) with fuming HNO3 and H2SO4 in 26% yield, then reduced to the amino compound (35) with NH2NH2 and Pd/C (81%). The free amine was reacted with succinic anhydride to give the amide-acid in 59% yield. EDC-HCl coupling of the amide-acid to the serinol proceeded in 63% yield. Subsequent DMT protection (51% yield) and phosphoramidation (82% yield) gave the desired building block 2 (Fig. 2).

Compound 4 (serinol–phenanthroline phosphoramidite building block)
Phenanthroline was nitrated (36) in fuming H2SO4 and HNO3 in 46% yield, then reduced in a similar fashion (35) with NH2NH2 and Pd/C in 85% yield. Reaction with succinic anhydride (72%), EDC-HCl coupling (60%), DMT protection (64%) and phosphoramidation (73%) yielded the building block 4 (Fig. 2).

Compound 5 (serinol–6,6''-dimethyl-terpy phosphoramidite building block)
6-Methyl-2-pyridinecarboxaldehyde was oxidized with sodium chlorite and hydrogen peroxide in phosphate buffer (37) (100% yield). The corresponding acid was then esterified with SOCl2 and ethanol (100% yield). The ester derivative was converted to the trione (38) in a 40% yield, and cyclization with NH4OAc proceeded in 76% yield. Chlorination with POCl3 and PCl5 produced the 4'-chloro-6,6''-terpy derivative (33%). Nucleophilic aromatic substitution for 4-hydroxybutyric acid yielded the free acid (88%). EDC-HCl coupling to serinol (73%), DMT protection (62%) and phosphoramidation (76%) yielded the building block 5 (Fig. 2).

Preparation of the RNA
The 159mer RNA substrate was synthesized by runoff transcription using Ambion’s MEGAshortscriptTM T7 kit and 5'-end labeled with Ambion’s KinaseMAXTM kit. The 28mer substrate was purchased (Oligos Etc.) and 5'-end labeled in a similar fashion. Both were purified by excision from a high-resolution polyacrylamide gel.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Observations which led to the molecular design employed
This report represents the next major enhancement to our ribozyme designs, which came about through the influence of four factors. (i) The pioneering work of Sigman and co-workers (8) on the oxidative cleavage of DNA by phenanthroline and its derivatives. (ii) Solution-state dimerization of the ribozyme mimics that contain an embedded terpyridine residue was observed using direct-infusion, negative-electrospray mass spectroscopy (Fig. 1). These µ-OH bridged dimers are notorious in Cu-terpyridine chemistry; furthermore, they are inactive at RNA transesterification. (iii) Ribozyme mimics constructed containing multiple terpyridine moieties were completely inactive for RNA transesterification. It is suspected that an inactive intramolecular-Cu(II) dimer was being formed (J.K.Bashkin, E.I.Frolova, A.T.Daniher, B.N.Trawick, unpublished results, see Fig. 3). (iv) Linkletter and Chin (27) reported greatly enhanced RNA transesterification by Cu(II) complexes when they employed the neocuproine ligand that suppressed the formation of Cu(II) dimers [(bipy) Cu (µ-OH)2 Cu (bipy)]2+ by steric interactions.



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  		Figure 3. A representation of intermolecular ribozyme mimic dimerization through [Cu(II) (µ-OH)2 Cu(II)]2+ centers supported by mass spectral studies.

 
The dramatic success achieved by Linkletter and Chin (27) is indicated by the pseudo-first-order rate constants for the cleavage of RNA dimer ApA obtained with Cu(II)–terpy, Cu(II)–bipyridine and Cu(II)–neocuproine: 1.9 x10–5 s–1, 1.9 x 10–7 s–1 and 3.9 x10–3 s–1, respectively. The Cu(II)–neocuproine complex cleaved RNA between two and four orders of magnitude faster than the closely related terpy and bipy complexes.

Although results derived from RNA dimers are not always directly applicable to the cleavage of polymeric RNA substrates (i.e. mRNA) (31), we believed that a strong correlation could be made between Chin’s creative results with Cu-mediated ApA cleavage (27) and our observations with macromolecular ribozyme mimics. Here we report the direct mapping of the optimization of small-molecule catalysts onto a macromolecular scaffold. We approached the problem by preparing ribozyme mimics based on terpy, 6,6''-dimethyl-terpy (dimethylterpy), o-phen and 2,9-dimethyl-o-phen (neocuproine). We also used a control sequence with no pendant ligand, but containing an abasic site.

Synthesis of phosphoramidite building blocks
Probe 1, which was synthesized from the commercially available phosphoramidite building block 1, is a control probe to test for any site-specific cleavage derived solely from the presence of an abasic site (33). Phosphoramidite 3 is the previously reported serinol–terpyridine building block and was prepared as before (Fig. 2) (33). Three novel phosphoramidite building blocks derived from serinol (phosphoramidites 2, 4 and 5) were prepared. The syntheses of compounds 2 and 4 are shown in Scheme 01, and synthesis of phosphoramidite 5 is detailed in Scheme 02.



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  		Figure 2. Phosphoramidite building blocks 1–5 used to prepare probes 1–5.

 


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  		Scheme 1. Synthesis of o-phen- and neocuproine-derived phosphoramidite building blocks. Phenanthroline (R = H). (a) fum. H2SO4, HNO3, 170°C, 1 h, 46%. (b) NH2NH2, Pd/C, EtOH, reflux, 5 h, 85%. (c) Succinic acid, pyridine, room temperature, 12 h, 72%. (d) EDC-HCl, serinol, DMF, room temperature, 12 h, 60%. (e) DMT-Cl, pyridine, room temperature, 12 h, 64%. (f) 2-Cyanoethyl N,N-diisopropylphosphoramidite, room temperature, 1 h, 73%. Neocuproine (R = CH3). (a) H2SO4, fum. HNO3, reflux, 5 h. (b) NH2NH2, Pd/C, EtOH, reflux, 5 h, 26% yield two steps. (c) Succinic acid, pyridine, room temperature, 12 h, 59% yield. (d) EDC-HCl, serinol, DMF, room temperature, 12 h. (e) DMT-Cl, pyridine, room temperature, 12 h, 49% yield two steps. (f) 2-Cyanoethyl N,N-diisopropylphosphoramidite, room temperature, 1 h, 82% yield.

 


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  		Scheme 2. Synthesis of dimethylterpy-derived phosphoramidite building blocks. 6,6''-Dimethyl-terpy: (a) NaClO2, NaH2PO4, H2O2, 10°C, 4 h, 100%. (b) SOCl2, EtOH, reflux, 6 h, 100%. (c) Acetone, NaH, THF, reflux, 6 h, 40%. (d) NH4OAc, EtOH, reflux, 6 h, 76%. (e) POCl3, PCl5, reflux, 12 h, 33%. (f) 4-Hydroxybutyric acid, KOH, DMSO, 55°C, 24 h, 88%. (g) EDC-HCl, HOBt, serinol, DMF, 12 h, room temperature, 73%. (h) DMT-Cl, pyridine, room temperature, 12 h, 62%. (i) 2-Cyanoethyl N,N-diisopropylphosphoramidite, CH2Cl2, 1 h, room temperature, 76%.

 
Synthesis of ribozyme mimics
These building blocks were incorporated at the 11th position of a 17mer DNA strand by standard automated DNA synthesis. The family of sequences used is detailed here: 5'-CTA CAT AGT CXC TAA AG-3', where X = three-carbon linker (probe 1) [from phosphoramidite 1]; serinol–neocuproine (probe 2) [from phosphoramidite 2]; serinol–terpyridine (probe 3) [from phosphoramidite 3]; serinol–phenanthroline (probe 4) [from phosphoramidite 4]; serinol–dimethylterpy (probe 5) [from phosphoramidite 5].

These probes were designed to target a 17mer region that is common to a 28mer RNA and to a 159mer fragment of the HIV gag gene mRNA. The 28mer RNA sequence is 5'-AAACCAACCCUUUAGAGACUAUGUAGAC-3' (recognition sequence underlined). The full sequence of the 159mer was published previously (15).

RNA cleavage
Figure 4 is a representative 20% polacrylamide denaturing gel (8 M urea) of the cleavage of the 28mer RNA by the different probes when incubated with copper. Cleavage reactions were conducted with 5'-end labeled RNA at a concentration of ~10–7 M. Each reaction contained 5 µM probe, 5 µM Cu(SO4), 0.1 M NaClO4 and 10 mM HEPES (pH 7.4) in a total reaction volume of 10 µl at 37°C for 15 h. Cleavage of the 159mer (Fig. 5) was carried out under similar conditions, but with an RNA concentration of ~10–8 M. Two reactions were conducted with each probe: (i) in the absence of added metal and (ii) in the presence of added Cu(II)SO4. EDTA (10 µM) was added to the control reaction of probe 3 as described previously (33) to remove trace Cu(II) scavenged from the buffers that arises due to the very strong binding of terpy for Cu(II). The extent of site-specific cleavage (for the reaction times noted) in the presence of Cu(II) is reported in Table 1.



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  		Figure 4. Autoradiogram of a 20% polyacrylamide gel (8 M urea) of the site-specific cleavage of the 5'-end-labeled 28mer RNA (0.1 µM) by ribozyme mimics (reaction conditions: 15 h, 37°C). Lane 1, unreacted RNA starting material; lane 2, ribonuclease T1 digest (G specific); lane 3, NaOH digest; lanes 4 and 5, reaction with no ribozyme mimic present both in the absence and presence of copper; lanes 6 and 7, treatment with probe 1 [three-carbon linker (5 µM)] both in the absence and presence of copper; lanes 8 and 9, treatment with probe 2 [11.NEO (5 µM)] both in the absence and presence of copper; lanes 10 and 11, treatment with probe 3 [11.ST (5 µM)] both in the absence and presence of copper (EDTA treatment to remove trace contamination of copper); lanes 12 and 13, treatment with probe 4 [11.PHE (5 µM)] both in the absence and presence of copper.

 


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  		Figure 5. Audioradiogram of 6% polyacrylamide gel (8 M urea) of the site-specific cleavage of the 159mer RNA (0.01 µM) by ribozyme mimics (reaction conditions: 10 h, 37°C). Lane 1, unreacted RNA starting material; lane 2, ribonuclease T1 digest (G specific); lane 3, NaOH digest; lanes 4 and 5, treatment with probe 2 [11.NEO (5 µM)] both in the absence and presence of copper; lanes 6 and 7, treatment with probe 3 [11.ST (5 µM)] both in the absence and presence of copper (EDTA treatment to remove trace contamination of copper); lanes 8 and 9, treatment with probe 4 [11.PHE (5 µM)] both in the absence and presence of copper.

 

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  		Table 1. Sequence-specific cleavage of 28mer and 159mer RNA targets by ribozyme mimics derived from Cu(II) and probes 1–5
 
The major site of cleavage varies by one base for o-phen- and terpy-derived catalysts. For example, with the 28mer RNA substrate, probe 3 cleaves to the 3'-side of A16, whereas probes 2 and 4 both cleave at the 3'-side of G15. A parallel variation in the site of cleavage is seen with of the 159mer substrate (Table 1). The cleavage products from the transesterification/hydrolysis reactions carried out by the ribozyme mimics co-migrate with both the NaOH digest and the RNase T1 digest, as can be observed in Figure 4, a high-resolution gel for the cleavage of the 28mer. RNase T1 cleaves RNA at G residues, leaving a guanosine-3'-monophosphate terminus and a 5'-OH terminus. Furthermore, the zinc(II) adduct of probe 3 produces the same cleavage product as the Cu(II) derivative (39). Zinc is a metal that has no accessible oxidative cleavage pathway under our reaction conditions. For every RNA cleavage reaction, background cleavage was found at all of the 5'-UA-3' sites, in keeping with their high natural propensity for scission (17,40,41).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY MATERIAL
 REFERENCES
 
The enhancement in activity noted for the presence of alpha methyl groups in the case of the phenanthroline ring system (probe 4 versus probe 2) is not found in the terpy system (probe 3 versus probe 5). The dimethylterpy-derived probes may be too sterically hindered to be active.

The extent of cleavage reported here follows the trends that Linkletter and Chin (27) observed for the parent metal complexes. The ordering of the ribozyme mimics in terms of transesterification efficiency is: probe 2 (neocuproine) >> probe 3 (terpy) > probe 4 (o-phen) >>> probe 5 (dimethylterpy-inactive) = probe 1 (abasic control-inactive)

The reactivity trend is strongly influenced by the pKa values of water coordinated to the copper centers [pKa values: Cu(neocuproine) 7.0 and Cu(terpy) 8.08]. Cu(II)–neocuproine has a double advantage as a RNA transesterification agent. Under our reaction conditions, the concentration of Cu(II)-OH (the most probable active species) is much greater for neocuproine than for terpy or o-phen, and the methyl groups of neocuproine suppress the formation of µ-OH bridged dimers. Examining the speciation of Cu(II)–terpy with respect to the pKa, a ratio of 17:83 of Cu(II)-OH to Cu(II)-OH2 is expected, and with Cu(II)–neocuproine a ratio of 72:28 is expected at pH 7.4. Assuming the metal-bound hydroxide is the active species, an enhancement of 4.2-fold should be found. In both the 159mer (10-fold) and the 28mer (4.6-fold) a greater enhancement is observed. These results demonstrate the double advantage of neocuproine.

We conclude that the high concentration of Cu(II)-OH and the suppression of hydroxide-bridged dimers by neocuproine allow Cu(II) to better reach its catalytic potential as an RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material, including characterization of novel compounds, is available at NAR Online.


   ACKNOWLEDGEMENTS
 
We thank Dr Lee Ratner for the plasmid containing the HIV gag-gene fragment. Acknowledgment for partial support of this research is made to the donors of The Petroleum Research Fund, administered by the ACS, and to NSF grant CHE-9802660. Mass spectrometry was provided by the Washington University Mass Spectrometry Resource, an NIH Research Resource (Grant No. P41RR0954). W.C.P. was an NSF-GOALI fellow at Reliable Biopharmaceutical Company in 1998. B.N.T. was a Chancellor’s Graduate Fellow (1995–2000).


   FOOTNOTES
 
* To whom correspondence should be addressed at present address: Pharmacia Corporation R3A, 800 North Lindbergh Boulevard, St Louis, MO 63167, USA. Tel: +1 314 694 3244; Fax: +1 314 694 3479; Email: james.k.bashkin{at}pharmacia.com Present addresses:William C. Putnam, Midwest Research Institute, 425 Volker Boulevard, Kansas City, MO 64110, USABobby N. Trawick, MetaPhore Pharmaceuticals, Inc., 1910 Innerbelt Business Center Drive, St Louis, MO 63114, USA Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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