Indian Journal of Chemistry Vol. 41A, January 2002, pp.65-72

An inorganic approach to drug design: Novel inorganic nucleases t

J A Cowan

Evans Laboratory of Chemistry, The Ohio State University, 100 West ISth Avenue, Columbus, Ohio 43210, USA

Received 25 July 2001

Copper aminoglycosides are demonstrated to be highly efficient cleavage catalysts for DNA and RNA targets. Such catalysts mediate both oxidative and hydrolytic pathways and their cleavage reactions display enzyme-like Michaehs­Menten kinetic behaviour. An unusual double-strand cleavage of DNA has been observed. Degradation of RNA viral motifs have been demonstrated in vitro, and the efficacy of such molecules against an ill vivo target has also been established using a novel in vivo assay. Chemical mechanisms underlying the cleavage of RNA and DNA targets, recognition strategies, mechanisms of cellular delivery, and the design of ill vivo assays have been presented.

Introduction There is considerable interest in the design, synthesis and characterization of molecules that target RNA or DNA motifs; either to inhibit the binding of cognate proteins or enzymes, or to mediate strand scission. Molecules in this category are diverse, and include organic dyes and cations, inorganic metal complexes, and natural antibiotics J

-8

. Since oxidative cleavage is mediated by reactive oxygen species (ROS) that are diffusible and can cause other severe cytotoxic effects, drug-mediated cleavage pathways for nucleic acids by phosphate ester hydrolysis are preferred. The phosphodiester linkage is, however, among the most inert chemical functional groups toward hydrolysis and this important feature has been utilized by Nature in the design of the structural framework for DNA and RNA9

.

Hydrolytic degradation of nucleic acids by nuclease enzymes is a critical biological reaction and metal ions play a central role in mediating such cleavage pathways. Hydrolysis of DNA by small molecules is primarily hindered by the repUlsive interaction of the negatively-charged phosphate group toward an incoming nucleophile. However, various transition metal and lanthanide cations have been shown to alleviate this repulsive interaction by direct

t Contribution from Evans Laboratory of Chemistry, The Ohio State University, 100 West IS'h Avenue, Columbus, Ohio 43210 t Based on a Lecture Presented at the International Symposium on Advances in Bioinorganic Chemistry held at TIFR, Mumbai, India in November 2000 t This work was supported by grants from the Petroleum Research Fund, administered by the American Chemical Society, The American Foundation for AIDS Research, and the National Science Foundation, CHE-Oll1161.

inner-sphere binding of the phosphate ester. Inorganic complexes that cleave DNA and RNA in a sequence specific manner are of potential value in the treatment of cancer and viral diseases, and for application in biotechnology 10.

In this review I focus on a new family of copper aminoglycosides that show up to two orders of magnitude greater activity than those for other synthetic metallonucleases and show great promise for the development of novel drugs and footprinting agents. The high level of activity of these complexes most likely reflects the higher binding affinity of the positively-charged aminoglycoside ligands to DNA and RNA relative to other neutral or less posi tively­charged ligands . A recent report from our laboratory has summarily compared the activities of published metallonucleases and detailed the proper procedure for kinetic evaluation of the activities of these complexes II. In spite of major advances in the field, a major limitation stems from the relatively low cleavage efficiencies (after correction for catalyst concentration) and the absence of multi-turnover kinetic behaviour. We hypothesized that significant improvements could be made through use of a catalyst that demonstrated a higher binding affinity to the substrate, while retaining a reactive metal center. The positively-charged family of aminoglycosides allowed both of these criteria to be met. High positive charge yields tight binding to negatively-charged nucleic acid substrates, while aminoglycosides react readily to form complexes with transition metal ions. Moreover, such ligands have been shown to bi!'ld selectively to structured RNA, motifs (such as those in Fig. 1)12-1 5, and offered us the promise of

66 INDIAN J. CHEM., SEC A, JANUARY 2002

Fig. I-Structure of the human hepatiti s delta virus ribozyme. (From ref. 32).

developing efficient reagents (metalloamino­glycosides) against viral targets. In fact our Cu2+_ neamine complex showed kobs (or VOlax ) of around 0.031 min-I at the highest concentration employed, thereby enhancing the rate of DNA hydrolysis by 5.2 x 107 times. The rate is approximately twice as efficient as that for the binuclear C03+ complex reported by Schneider's groupl6, although the plasmid employed, catalyst concentration, and the pH are not necessarily the same. The specificity constants (kca/ KM) give the hydrolysis rate enhancement per micromolar catalyst concentration and reach a value of 4.8 x 105 h-I M IO for Cu2+-neamine under pseudo­Michaelis-Menten conditions, which is two orders of magnitude greater than those seen for various metal ions, including lanthanides.

Synthesis and characterization of metalJoaminoglycosides

The synthesis of the key compounds Cu2+_ (kanamycin) 1 and Cu2+-(neamine), 2, have been , , ." described by us In recent publIshed work .

Characterization of these compounds by I3C relaxation experiments indicates coordination as shown, while the pH dependence of the relaxation data supports coordination of an alkoxide ligand with loss of the hydroxyl proton". Compound 1 has previously been reported in a different context l7

, 18 and the metal coordination sites confirmed by acetylation and I3C relaxation measurements. The structures for 1 and 2, shown below, can be taken as a reli ab le indicator of the molecular architecture of the molecule. All metalloaminoglycosides obtained in our hands have been purified and give satisfactory elemental analysis, EPR, and UV -vis data ".

Hydrolytic DNA cleavage by Cu2+-(kan A) and Cu2+_ (neamine)

Both 1 and 2 were found to rapid ly degrade supercoiled (form I) plasmid DNA (Fig. 2) under hydrolytic conditions in the absence of added redox cofactors. A systematic reactivity profile of the isolated and purified Cu-(kan A) complex with plasmid allowed observation of nicked DNA (form II) after brief (5 min) exposure to 0.5 /lM complex . DNA treated with 1 /lM Cu2+(aq) or 5 /lM aminoglycoside (kanamycin A or neamine) did not show evidence of cleavage when incubated at 37°C for 60 to 120 min. DNA cleavage by Cu-(kan A) was completely inhibited when the solution was saturated with metal­free kanamycin A (5 /lM) or NaCI (150 mM), consistent ' with the importance of electrostatic interactions in the binding of the drug to DNA. UV­Vis and EPR spectroscopic characterization carried out under the ionic conditions employed in cleavage experiments (NaCI = 150 mM) demonstrate these complexes to be stable over the time-frame of cleavage studies.

Plots of the appearance of form II or the disappearance of form I versus time followed pseudo­first-order kinetic profiles and fit well a single exponential decay curve. DNA Cleavage reactions were monitored under Michaelis-Menten kinetic

------- -1 2 3 4 5 678 Lanes

Fig. 2-Cleavage of supercoiled plasmid DNA by metal kanamycin A derivatives. Lanes 1, DNA + 5 iJ.M kan A; 2, + y­(kan A); 3, Fe-(kan A) ; 4, Mn-(kan A) ; 5, Co-(kan A); 6, Ni-(kan A); 7, Cu-(kan A); 8, Zn-(kan A). Each reaction mixture contained 5 I.! iJ.M DNA (base pair concentration), ! iJ.M metal kanamycin A complex and were incubated for ! h at 37°C. Similar over-degradation of DNA was observed fo r Cu-(neamine) and Cu-(kan A) (lane 7).

COWAN: NOVEL INORGANIC NUCLEASES 67

conditions using a constant catalyst concentration (l00 1lM) and varying substrate concentration (20 to 200 1lM) (Fig. 3). Under these conditions V max (0.0595 min-I), k~at (5 .95 X 104 min-I) and KM (4l.6 1lM) values were determined, and so the complex appears to bind to the plasmid in an enzyme-like manner.

The rate enhancements under these conditions are the largest reported yet for any metal complex (after revision of published data to eliminate differences arising from definition of concentrations) and probably reflect the large binding constants for Cu2

+_

neamine to plasmid DNA (-2.6 x 105 MI). In fact, our preliminary results demonstrate the rate enhancement for Cu2

+ -(kan A) cleavage to be even greater. We have shown previously that neomycin Band kanamycin A bind nucleic acid components with affinities in the range of 3.9 x 103 to 7.0 X 104 MI (Ref. 19) Accordingly, while the aminoglycoside ligand provides tight binding to the negatively­charged phosphate backbone in DNA, the metal center mediates phosphodiester cleavage.

Demonstration of hydrolytic cleavage in the absence of electron donors

We have characterized the phosphorylation state of

0.05

I" 0.04

c 'E 0.03

VI .D 0 ~ 0.02

0.01

0.00

o

•

•

----..... ------.. 12345678bne

50 100 150 200

[DNA]~

Fig. 3-Saturation kinetics for the cleavage of plasmid DNA using 100 /lM Cu2+-neamine. Varying concentrations of plasmid were reacted with constant concentration of the drug for 120 minutes at 37°C in 10 mM HEPES, pH 7.30 (see inset). Samples were run on a 1% agarose gel and stained with ethidium bromide (see inset) . Inset: Cleavage profile for varying concentrations of plasmid DNA showing nicked (upper band) and supercoiled (lower band). Lanes I, DNA (20 /lM); 2, 20 /lM DNA; 3, 36.3 /lM DNA; 4, 48.4 /lM DNA; 5, 72.6 /lM DNA; 6, 84.7 /lM DNA; 7, 96.8 /lM DNA; 8, 193.6 /lM DNA. Samples in lanes 2-8 were incubated in the presence of 100 /lM Cu2+-neamine, as described above.

the ends generated following hydrolysis by Cu­aminoglycoside treatment in a series of experiments using T4 PNK (polynucleotide kinase) and TdT (terminal deoxynucleotide transferase). T4 PNK specifically requires a free 5' -OH group to add the y­phosphate of ATP. Should the 5' -end be phosphorylated, labeling is only possible after prior treatment with calf intestinal alkaline phosphatase (ClAP) to generate a free 5' -OH group. In turn, TdT adds an a-phosphate from dNTPs only to the 3 ' ­hydroxyl groups of DNA. Following hydrolytic cleavage by Cu-neamine, the plasmid can have either a 5'-phosphate or a 5'-hydroxyl group. Reaction of the Cu-neamine-treated plasmid with T4 PNK under "forward reaction" conditions did not yield a signal when the sample was analyzed by autoradiography following gel electrophoresis (Fig. 4). However, when treated with ClAP first (to dephosphorylate the 5'-end to generate 5'-OH) and then carrying out the forward T4 PNK reaction, we could quantitatively measure y-32p incorporation into the product DNA fragments (Fig. 4). This indicates that the DNA possesses a 5'­phosphate following treatment with Cu-neamine. Further support of such a hydrolytic mechanism, yielding 5' -phosphate and 3' -OH terminae was provided when we treated the DNA with [a_32PJ dGTP and TdT. TdT is a template-independent polymerase that catalyzes the incorporation of [cx.-32PJ _dG only when the 3'-end of DNA has a free hydroxyl group". As shown in Fig. 4, [cx._32p] was readily incorporated into the Cu-neamine-treated DNA. Both the necessity to dephosphorylate before incorporation of 5' -phosphate using PNK, and incorporation of [cx.-32PJ-dG, clear! y demonstrate the generation of both 5'-phosphate and 3'-hydroxyl

324 Lanes

Labgl~DNA - ••

32 Free P NTP

Fig. 4- Agarose gel electrophoresis of Cu-neamine-treated pT7-7 after incubation with T4 PNK and [y_32 P] ATP under conditions favouring the: (I) exchange reaction; (2) forward reaction; and (3) after ClAP treatment. Lane (4) shows Cu-neamine-treated pT7-7 following incubation with TdT and [a}2 P] dGTP.

68 INDIAN J CHEM. SEC. A, JANUARY 2002

groups in pT7-7 when treated with copper aminoglycosides (Fig. 4). Moreover, high resolution polyacrylamide gel analysis of products generated from DNA upon copper-aminoglycoside treatment reveals co-migration with phosphorylated oligo (dT)4-22 fragments. No piperidine or aniline treatment (usually required for oxidative cleavage and base loss) was required to generate fragments before analysis by gel electrophoresis experiments2o. In fact by use of HPLC assays we have found that base release for DNA cleavage is achieved only under oxidative conditions, where either ascorbic acid or H20 2 was used in concert with Cu-aminoglycosides.

Targeting RNA structures Aminoglycosides antibiotics do in fact constitute a

large family of molecules that find extensive clinical use in the treatment of gram-negative infections21

.

The antibacterial activity of these molecules has been attributed to binding to ribosomal RNA with inhibition of protein translation. Recent reports have also demonstrated selective and high affinity binding of aminoglycosides to a variety of other RNA structural motifs22

• Molecular recognition of nucleic acids can arise by virtue of binding specificity at the level of primary and secondary structure; however; it

c

'~lJ cu

~ cc CC

C0> 5' 3'

R23

6' ~H, RNA recognition

HO~~

HO~HO H,N HOH,W:~NH, D reI

HO H,N "H NH 0 OH

6" .... ' ,

~ available for neomycin B metal binding

is at the level of tertiary structure that the highest level of selectivity with non-nucleotide ligands should be achieved. Retroviruses such as RNA tumor viruses and HIV contain single strands of RNA that are folded into a variety of secondary and tertiary conformations, including local regions of duplex structure, some of which may be distorted due to base mismatches, bulges, pseudoknots and hairpins. Similar secondary and tertiary folding patterns are common for rRNA and mRNA from all sources. Few non-nucleotide ligands possess secondary structural specificity; however, recognition at the level of tertiary structure is particularly relevant for structured RNA motifs . Compounds capable of specific binding to RNA over DNA are attractive targets for development of anti-viral drugs (Fig. 1).

Hydrolytic cleavage chemistry with RNA

With DNA we have demonstrated efficient nucleic acid cleavage chemistry to be mediated by copper aminoglycoside complexes. In as much as the aminoglycoside ligands provide molecular recognition and tight binding to a target RNA sequence, efficient catalytic RNA cleavage chemistry at physiological pH and temperature was also expected and has in fact been demonstrated in our

R23:neomycin B complex

ring B

A 13

Fig. S-Illustration of the complex of R23 and neomycin B. In the schematic inset at top left, the binding domain of R23 is boxed. Base AI3 flips out of the loop and appears to form a latch across the groove where the aminoglycoside sits. This geometry is defined by 20 NOEs between the base ring and bound neomycin B 19.

COW AN : NOVEL INORGANIC NUCLEASES 69

laboratory23. An RNA aptamer with high binding affinity for neomycin B has been reported and a modified 23-mer RNA (hereafter termed R23) with additional GC base pairs at the base of the stem (Fig. 5) was selected for a preliminary investigation of this approach23. The solution structure of the neomycin complex with R23 has been determined by NMR in our laborator/9 and is similar to that for a related sequence solved by Patel and coworkers24. Both structures show that the aminoglycoside binds to the major groove of R23 through electrostatic and hydrogen bonding interactions with the amine and hydroxyl groups of rings A and B of the aminoglycoside, while the C and D sugar rings are pendant and can potentially chelate metal ions (Fig. 5). The kanamycin A ligand of structure 2 lacks the ribose ring C and two amine groups in ring D of neomycin B (Fig. 5); however, neither ring contributes significantly to binding l9,24, and so it is presumed that kanamycin A and neomycin B bind in a similar manner. In fact, a comparison with published data indicates that rings A and B appear to form a conserved structural motif for recognition and binding to ribosomal RNA25

,.26. The aminoglycoside binds to the major groove and there exists an extensive hydrogen bonding network from rings A and B, and significant salt bridge interactions from the amines in ring D to backbone phosphates (Fig. 5). Rings A and B (the neamine fragment) appear to form the critical hydrogen bonding contacts with the RNA. This work has been published by US

I9 . Experimental gel electrophoretic studies of the

cleavage products of end-labeled and body-labeled R23 show evidence of efficient cleavage chemistry under mild reaction conditions23. Under hydrolytic conditions, two sites of cleavage could be identified: one in the loop region at G15, and the other in the stem region at C4, consistent with the NMR solution structure of R23 RNA bound to neomycin B, which shows binding of the aminoglycoside antibiotic in the loop region A 13 A 14G15, and in the step region U5G6G7GS (ref. 19).

Oxidative c1eavagechemistry with RNA Although the hydrolysis of RNA by 1 is

appreciable, addition of redox agents such as ascorbic acid/02 or H20 2 increases the cleavage efficiency dramatically. Addition of 100 ~ ascorbic acid to R23 treated with 20 nM 1 shows extensive cleavage with almost 100% digestion of the parent RNA. Ascorbate + Cu2+ alone caused no cleavage (Fig. 6,

lane 6). We have demonstrated that 1 mediates oxidative cleavage of DNA through reactive copper­oxo or copper-hydroxo species27. While 1 catalyzes the formation of hydroxy radicals in the presence of H20 2 or ascorbate, causing oxidative damage to nucleic acids27

, such hydroxyl radicals could not be trapped in the presence of a nucleic acid substrate. This indicates that the radicals formed are localized on or close to the copper ion and are non-diffusible. Thus the products from reaction of 1 with R23 RNA in the presence of H20 2 or ascorbic acid are indicative of an oxidative mechanism, mediated by a copper redox couple.

A primary reason for cleavage efficiency at low complex concentration arises from the high binding affinity for an R23 target site. No cleavage products were observed after treating R23 with comparable concentrations of either Cu2+(aq) or metal-free aminoglycoside alone, while control experiments with linear 0Iigo(A I2.ls) or poly(C), which bind aminoglycosides very weakly, also gave no cleavage under similar reaction conditions. Also, conditions of high ionic strength (> 150 mM NaCl) and addition of metal free kanamycin were found to inhibit cleavage of R23 with 1. These results demonstrate the requirement for targeted binding to effect cleavage. Similar cleavage efficiencies for ds-DNA were observed only at much higher complex concentrations (0.1 to 0.5 ~ as a result of the significantly lower binding affinity relative to a cognate structured RNA motie7

•

RRE cleavage

Productive HIV infection is dependent on the interaction of a regulatory protein, Rev, with a specific RNA structure known as the Rev-responsive element or RRE. The RRE is a 234 nucleotide RNA sequence embedded within the viral env coding region. The high affinity Rev binding site, or core element, within the RRE consists of a stem-bulge­stem structure (a 67-mer). Since the RRE-Rev interaction is essential for viral replication, it is an attracti ve target for anti viral therapy. Several aminoglycosides, especially neomycin B, have been demonstrated to selectively bind RRE2S.29. The RNA cleavage chemistry of copper aminoglycoside complexes was studied using either body-labeled, or 5,-32P-end-Iabeled 67-nt RRE in the presence and absence of reducing agents such as ascorbic acid, hydrogen peroxide or hydroxylamine hydrochloride. RRE was incubated at 37°C for 90 min to 2 h with

70 INDIAN 1. CHEM., SEC A, JANUARY 2002

Lane

1234567 , , - -

. -.. - _~G15 -.. ' .. • -.G1O

U. _~G8

I _~G7

_~G6

8 •

Fig. 6-Autoradiograph of a 19% denaturing polyacrylamide electrophoretic gel for the cleavage of uniformly-labeled R23 RNA by 1 at 37 °c for I hr. Lane I, R23; lane 2, R23 + 0.1 M NaOH; lane 3, R23 + Na2COiNaHC03 buffer; lane 4, R23 +0.25 units RNase TI ; lane 5, R23 + 0.5 units RNase TI; lane 6, R23 +

111M CuS04/100 11M ascorbate; lane 7, R23 + 20 nM 1/100 11M ascorbate. Cleavage reactions were carried out in Tri s-HCI (20

mM; pH 7.5) and contain ca. 10 nM RNA. Captions on the right indicate G-specific cleavage by RNase TI in lanes 4 and 5.

various concentrations of the metal complexes, and the products of cleavage were separated on a 15% denaturing polyacrylamide-sequencing gel (Fig. 7). Control reactions in the presence of metal free aminoglycosides and CUS04 alone were carried out and the products were analyzed by PAGE. Cleavage of RRE (5 11M) was observed with as low as 1 /lM concen tration of Cu2

+ -kanamycin A (Fig. 7) and 100% cleavage was observed using 1 mM complex (significant cleavage is also observed in Fig. 8 with 0.5 mM complex). Cleavage sites were assigned by comparison with products generated from an RNase Tl generated G-specific ladder (Fig. 7, lane 5) and partial alkaline hydrolysis (Fig .. 7, lane 6).

1 ~3456

.. . . . . " 1':

..

•

Fig. 7-Hydrolytic cleavage of 5' } 2P-RRE by Cu2+-kanamycin

A. Lanes: (I) RRE (-1 lAM); (2) RRE H 11M) + 5

IlMkanamycin A; (3) RRE (-1 I1M)+ 1 11M Cu2+-kanamycin A;

(4) RRE (-I'IAM) + 5 IlMCu2+; (5) RNase T 1: (6) partial alkaline

hydrolysi s of RRE (-111M). RRE was incubated with Cu2+_

kanamycin A in HEPES buffer, pH 7.3 for 90 minutes at 37°C. Reaction products were separated on a 15% PAGE after ethanol precipitation.

Data in Fig. 7 show that cleavage of labeled RRE by metal aminoglycosides is not random, but rather a specific cleavage site at G46 was observed (Fig. 7, lanes 3), resulting in the formation of a 5,_32p labeled 9-mer RNA fragment. The cleavage sites could be localized to the sites of drug binding at the internal loop where the metal-free aminoglycosides have been demonstrated to bind to RRE28. Highly selective RNA cleavage was observed, using body-labeled RRE, even under conditions of excess drug (Fig. 8). Any random cleavage would have resulted in a smear on the gel, however, distinct bands were obtained even when RNA:drug ratio was as high as 1 :200. Chemical footprinting analysis demonstrates that the drug interacts with a discrete RNA region, providing an explanation for specificit/8

. Within the RRE, both Rev and aminoglycoside binding sites overlap . Therefore, the inhibition of Rev-RRE by aminoglycosides is due to mutually exclusive interactions with a common set of bases [GGG( 46-48)). Higher concentrations of Rev protein can, however, overcome the inhibitory effects of aminoglycosides28

. Our observations of RRE cleavage at G46 of the GGG(46-48) bulge indicates that the

COW AN : NOVEL INORGANIC NUCLEASES 71

1 2 3 Lanes

~ -58-mer 57-mer

__ .-- 9-mer

Fig. 8-Cleavage of body-labeled 32p_ RRE. Lanes (I) RRE (S

f.LM) ; (2) RRE (S f.LM) + O.S mM Cu2+-kanamycin A; (3) RNA markers. Reaction products were separated on a 20% PAGE after ethanol precipitation and run against MW markers of specific size.

target RNA would be destroyed upon drug binding making it unavailable for Rev binding. It is remarkable that both Rev and neomycin B recognize a non-Watson-Crick homopurine base pair between G48:G71 and other non canonical interactions. This suggests that irregular RNA structures, especially bulges, are critical for aminoglycoside recognition and binding.

This result supports tight binding by the drug to RRE28

,29 with cleavage in a non-random manner. It is also clear from this gel that RRE cleavage by copper­kanamycin is hydrolytic in nature. Oxidative cleavage would have resulted in the loss of one nucleotide at the site of cleavage with the concomitant generation of a shorter RNA fragment. However, as seen in Fig. 8 (lane 2), cleavage results in bands corresponding to 58-mer and 9-mer product fragments, clearly supportive of a hydrolytic mechanism during RNA cleavage. Addition of co-reactants like ascorbic acid had no substantial effect on the cleavage of RRE by Cu2+-kanamycin. The products of such cleavage reactions co-migrate with those produced in absence of reductants. There was no change in cleavage efficiencies in the presence of either 1 mM or 2 mM

ascorbic acid under varying Cu2+-kanamycin concentrations, supporting our view that reductants do not play an important role in the cleavage of RRE. That is, we believe the oxidative path to be precluded for RRE, possibly reflecting the orientation of the reactive Cu-oxygen species relative to ribose ring C-H bonds. This contrasts with the cleavage of a distinct RNA aptamer (R23) that was described in the previous section23

.

Specificity of RNA cleavage by Cu-aminoglycosides The specificity of RNA cleavage by copper

aminoglycosides was probed using tRNAPhe. This

RNA has been extensively studied in the context of small molecule RNA cleavage agents30

. Reactions of tRNAPhe with various concentrations of copper aminoglycosides did not result in any cleavage as detected by polyacrylamide gel electrophoresis. Addition of 2 mM ascorbic acid to solutions containing tRNAPhe

, copper aminoglycosides (lOa ~ to 1 mM) also did not yield cleavage fragments when run on a 15% denaturing gel. Also, under similar experimental conditions, Cu2+ -kanamycin did not cleave linear poly (A), poly (C) or poly (G) RNA sequences23

,27 , For an RNA molecule possessing a tight binding site for aminoglycosides, the above conditions would have been sufficient to produce cleavage fragments. This selectivity of RNA cleavage by small molecules is unparalleled in the published literature. Typically, molecules that cleave RNA by antisense methodologies are based on RNA sequence31 rather than on its structure, and so copper aminoglycosides constitute the first examples of a new family of "inorganic ribozymes" that target structured viral RNA motifs. Both, this result and prior studies of an RNA aptamer,23 demonstrate that an important factor underlying the efficient cleavage chemistry shown by these molecules is the high binding affinity for the RNA target site.

Overview There is considerable interest in the design,

synthesis and characterization of molecules that target RNA or DNA motifs; either to inhibit the binding of cognate proteins or enzymes, or to mediate strand SCISSIOn. Our preliminary results demonstrate unprecedented RNA and DNA hydrolytic cleavage activities under physiological conditions. Low molecular weight inorganic complexes that cleave DNA and RNA in a sequence specific manner are of potential value in the treatment of cancer and viral diseases, and for application in biotechnology. We

I

72 INDIAN J. CHEM., SEC A, JANUARY 2002

have demonstrated copper aminoglycosides to be highly efficient cleavage catalysts for DNA and RNA targets. Such catalysts mediate both oxidative and hydrolytic pathways and their cleavage reactions display enzyme-like Michaelis-Menten kinetic behaviour. Further efforts will be required to expand our understanding of the chemical mechanisms underlying the cleavage of RNA and DNA targets and develop their use as bona fide inorganic drugs. Other requirements will be the development of in vivo assays, cell delivery mechanisms, and optimization of cleavage reagents for specific targets.

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