nihms122811.pdf

DNA Strand Cleavage Near a CC Mismatch Directed by a

Metalloinsertor

Mi Hee Lim

Irvin H. Lau

, and

Jacqueline K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,

California 91125

Abstract

Reagents for recognition and efficient cleavage of mismatched DNA without photoactivation were

designed. They contain a combination of a mismatch-directing metalloinsertor, [Rh

(bpy)

(chrysi)]

(chrysi = 5,6-chrysenequinone diimine), and an oxidative cleavage functionality,

[Cu(phen)

]

(

). Both unconjugated (

Rh+Cu

) and conjugated (

−

) frameworks of the Rh

insertor and

were prepared. Compared to

, both constructs

Rh+Cu

and

−

exhibit

efficient site-specific DNA scission only with mismatched DNA, confirmed by experiments

with

P-labeled oligonucleotides. Furthermore, these studies indicate that DNA cleavage occurs

near the mismatch in the minor groove and on both strands. Interestingly, the order of reactivity of

the three systems with a CC mismatch is

Rh+Cu

−

≫

. Rh binding appears to direct Cu

reactivity with or without tethering. These results illustrate advantages and disadvantages in

bifunctional conjugation.

Base pair mismatches in the genome can arise from damage by environmental agents (i.e. UV

light), a wide range of genotoxic chemicals as well as errors made by DNA polymerase during

synthesis.

These base mispairs are generally corrected by the mismatch repair machinery;

if they are not repaired, however, mutations arise, which subsequently lead to increased cancer

susceptibility.

–

To recognize DNA mismatches, we have developed rhodium complexes

containing an intercalating ligand that is too bulky to insert at stable matched sites and instead

preferentially binds to thermodynamically destabilized mismatched sites.

–

The complex [Rh

(bpy)

(chrysi)]

(Figure 1), designed by our laboratory, targets more than 80% of mismatches

with high selectivity and promotes single-stranded cleavage of the DNA backbone next to the

mismatch site upon photoactivation.

–

More interestingly, this Rh complex can detect and

photocleave a single base mismatch in a linearized 2725 base pair plasmid.

We have recently

elucidated how the complex [Rh(bpy)

(chrysi)]

interacts with the mismatched sites in DNA

by crystal structural determination and NMR analysis.

The binding mode of this complex

to the mismatch site is not classical intercalation but rather insertion. The expansive chrysi

ligand is deeply inserted into the mismatch site in the minor groove resulting in the complete

ejection of mismatched nucleotides from the base stack.

Recently, this mismatch-specific metalloinsertor has shown promise in cell-selective strategies

for chemotherapeutic design.

The Rh complex selectively inhibits cellular proliferation in

mismatch repair-deficient cells as compared to mismatch repair-proficient cells. In another

possible chemotherapeutic approach, bifunctional conjugates that combine a metalloinsertor

targeting DNA mismatches with a reactive species such as an aniline mustard or a cisplatin

*To whom correspondence should be addressed. Email: jkbarton@caltech.edu.

Supporting Information Available: Preparation of the conjugate

–

and Figures S1–S3. This material is available free of charge via

the Internet at http://pubs.acs.org.

NIH Public Access

Author Manuscript

Inorg Chem

. Author manuscript; available in PMC 2009 September 21.

Published in final edited form as:

Inorg Chem

. 2007 November 12; 46(23): 9528–9530. doi:10.1021/ic701598k.

NIH-PA Author Manuscript

analog have been designed.

–

Studies of these conjugates demonstrate that the Rh moiety

tunes the reactivity of the agent linked to the metalloinsertor.

Following this conjugation strategy, a construct can be devised for targeting and cleaving

mismatched DNA without light activation. To achieve this, we have investigated the reactivity

of a chemical DNA cleavage agent in the presence of [Rh(bpy)

(chrysi)]

. For efficient

oxidative cleavage, we have pursued the use of [Cu(phen)

]

with the Rh insertor. In the

presence of a reducing agent, the newly formed [Cu(phen)

]

(

, Figure 1) promotes light-

independent DNA backbone cleavage showing more localized cleavage sites than those

generated by [Fe(EDTA)]

−

.15

–

The complex

tethered to various DNA-binding moieties

exhibits efficient DNA scission.

The redox chemistry of [Cu(phen)

]

is feasible in the

reducing environment of living cells, suggesting also a potential biological application of the

copper species with the Rh insertor. Here we report the preparation of combined Rh and Cu

systems that are composed of the metalloinsertor and the Cu complex as well as an examination

of their DNA cleavage activity without photoactivation.

The reactivity of both nonconjugates (

Rh+Cu

) and conjugates (

−

) of [Rh

(bpy)

(chrysi)]

and

in DNA scission was examined. The Rh complex [Rh

(bpy)

(chrysi)]

Rh+Cu

(Figure 1) was synthesized as described.

For the conjugate

−

, we prepared its precursor [Rh(chrysi)(phen)(bphen)]

(bphen =

-[7-(4

′

-methyl-2,2

′

bipyridin-4-yl)heptyl]-

-(1,10-phenanthrolin-5-yl)pentanediamide) through the coupling

reaction of [Rh(chrysi)(phen)(bpy

)]

(bpy

= 7-(4

′

-methyl-2,2

′

-bipyridin-4-yl)heptan-1-

amine)

with the modified phen ligand (phen’ = 5-(1,10-phenanthrolin-5-ylamino)-5-

oxopentanoic acid),

as described in Scheme 1 and Supporting Information. Copper

coordination was accomplished

in situ

(

vide infra

), affording the conjugate

−

DNA cleavage experiments of

Rh+Cu

and

−

with 31mer oligonucleotides either lacking

or containing a CC mismatch were performed and monitored by denaturing polyacrylamide

gel electrophoresis (Figure 2). In the case of the reactions of

Rh+Cu

with DNA, duplex (1

M) was incubated with 1

M [Rh(bpy)

(chrysi)]

at 37 °C for 5 min, followed by addition

of 1

M CuCl

and 3

M phen; upon treatment with 5 mM ascorbate, the reagents were allowed

to react with DNA. After 5 or 10 min, the reactions were quenched by 5 min treatment with 5

mM dmp (dmp = 2,9-dimethyl-1,10-phenanthroline) followed by freezing. For the conjugate

−

, the Rh complex [Rh(chrysi)(phen)(bphen)]

M) was incubated in the following

order: duplex (1

M, 5 min, 37 °C), CuCl

M, 5 min, 37 °C), phen (3

M) and ascorbate

(5 mM). The reaction was then quenched in the same manner as described above. To compare

the reactivies of

Rh+Cu

and

−

was also generated

in situ

by reacting CuCl

M) with phen (3

M) followed by ascorbate (5 mM) and was similarly allowed to react with

duplex. As shown in Figure 2, site-specific DNA cleavage occurs only in the presence of

mismatched DNA, and the sites cleaved are near the mismatched site (two or three bases away).

No DNA cleavage without light is evident for the parent conjugate without

or any of the

individual reagents. More interestingly, the nonconjugate

Rh+Cu

exhibits more efficient DNA

scission than the conjugate

−

(cleavage yield

for 5 min = 35 and 19%; for 10 min =

65 and 41% by

Rh+Cu

and

−

, respectively, in Figure 2). Note that some cleavage is

evident also with

alone.

Binding of the Rh moiety of

Rh+Cu

and

−

to the mismatch

site was visualized independently by performing photoactivated DNA cleavage with [Rh

(bpy)

(chrysi)]

and [Rh(chrysi)(phen)(bphen)]

without

present (Figure S1). These

results together indicate that the recognition of mismatched DNA by the Rh moiety enhances

the cleavage activity of

close to the mismatch.

To obtain further structural information regarding the DNA cleavage site, we performed the

cleavage experiments using the

P-end-labeled complementary strands (Figures S2). Site-

specific DNA scission is again observed only with mismatched DNA.

On both strands, the

Lim et al.

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. Author manuscript; available in PMC 2009 September 21.

NIH-PA Author Manuscript

cleavage sites are mainly 2–3 bases from the mismatch toward the 3

′

-end (Figure 3).

Furthermore, more efficient DNA cleavage is found with the unconjugated framework

+Cu

than with the conjugate,

−

The 3

′

asymmetry in cleavage sites by

Rh+Cu

and

−

on both strands indicates that DNA

cleavage occurs in the minor groove (Figure 3).

This result is not surprising, since both the

parent Rh complex and Cu(phen)

are known to bind DNA from the minor groove side.

–

Thus both covalently and noncovalently, the Rh insertor directs reactivity of the Cu center

in the minor groove.

Since binding of the Rh insertor into the mismatch site results in ejection of the mismatched

bases, we also explored whether the Cu complex might react with the ejected bases. To evaluate

this, alkaline and piperidine treatment were performed after the reaction of the mismatched

DNA with

Rh+Cu

(Figure S3). No new scission products are observed, suggesting that the Cu

complex does not react with the bases or the ejected bases upon insertion of the Rh moiety in

mismatched DNA to form alkaline- or piperidine-sensitive lesions. Thus, the observed DNA

cleavage, induced by reaction of

, appears primarily with the sugar ring.

–

Why do we find preferential reaction with

Rh+Cu

rather than the tethered conjugate,

−

? One possible explanation is that Rh binding may locally distort the helix to facilitate Cu

(phen)

reaction. Reaction by Cu(phen)

is localized, directed by an intermediate high-valent

Cu(O) species.

Tethering to the Rh center may also somewhat confine the orientation of

the damaging agent. Additionally, tethering affects binding of the Rh complex within the

narrow minor groove. Measurements of the binding of [Rh(bpy)

(chrysi)]

and [Rh(chrysi)

(phen)-(bphen)]

by photocleavage assay (Figure S1) show a decrease in binding affinity to

the mismatched site associated with functionalizing the bpy ligand. Thus the relative

reactivities of

−

and

Rh+Cu

are likely a consequence of confinement of both the Rh and

Cu moieties in the particularly narrow minor groove of the DNA duplex.

This work establishes a new approach for developing an efficient cleavage agent without light

that is specific for mismatched DNA. In order to exploit the individual contributions of the Rh

and Cu complexes in recognition of mismatched DNA and strand cleavage, both species were

combined. This remains a useful strategy to couple recognition and reaction. Conjugation of

these two species, however, results in a reduction in both binding and reactivity of the

complexes with mismatched DNA. Indeed, for the CC mismatch, the parent Rh complex can

direct

reactivity without cumbersome synthesis. Although unexpected, these results

illustrate both the advantages and disadvantages of preparing bifunctional conjugates.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We are grateful to the NIH (GM33309) and Applied Biosystems for their financial support. We also thank the tobacco-

related disease research program (TRDRP) for a postdoctoral fellowship to M.H.L.

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14. A bifunctional conjugate has also been prepared with an unreactive fluorophore. See Zeglis BM,

Barton JK. J Am Chem Soc 2006;128:5654–5655.5655 [PubMed: 16637630]

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19. Sardesai NY, Lin SC, Zimmermann K, Barton JK. Bioconjugate Chem 1995;6:302–312.

20. Cleavage yield (%) = I

/(I

+ I

) × 100 (I

= intensity of the cleavage product bands; I

= intensity

of the parent strand band).

21. Cleavage with Cu(phen)

alone on the mismatched oligonucleotide may reflect dynamic fraying

associated with the destabilized mismatch.

22. Dervan PB. Science 1986;232:464–471. [PubMed: 2421408]

23. Thyagarajan S, Murthy NN, Narducci-Sarjeant AA, Karlin KD, Rokita SE. J Am Chem Soc

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Figure 1.

Chemical structures of

Rh+Cu

and

−

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Figure 2.

Sequences of 31mer oligonucleotides (top). Autoradiogram of a denaturing 20%

polyacrylamide gel presenting DNA cleavage by

Rh+Cu

and

−

with matched and

mismatched oligonucleotides (bottom). A+G and C+T, Maxam-Gilbert sequencing reactions.

* indicates 5

′

P-end-label of the strand. Conditions: duplex (1

M),

M CuCl

, 3

phen, 5 mM sodium ascorbate),

Rh+Cu

M [Rh(bpy)

(chrysi)]

, 1

M CuCl

, 3

M phen,

5 mM sodium ascorbate),

−

M [Rh(chrysi)(phen)(bphen)]

, 1

M CuCl

, 3

phen, 5 mM sodium ascorbate), in 10 mM Tris, pH 7.5, 50 mM NaCl at 37 oC. Lanes 1–2 and

7–8, fragments with

Rh+Cu

; lanes 3–4 and 9–10, fragments with

−

; lanes 5–6 and 11–

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12, fragment with

. The time shown reflects incubation time after treatment with sodium

ascorbate before quenching. The arrow marks the mismatched site.

Lim et al.

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Figure 3.

Representation of DNA cleavage sites by

Rh+Cu

(top) and

−

(bottom). The arrows

indicate the cleavage sites on the 5

′

P-end-labeled strands. The length of each arrow at the

given site reflects the relative percent cleavage on the strand. The mismatched site is

highlighted in red.

Lim et al.

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Scheme 1.

Preparation of

−

Lim et al.

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