nihms97864.pdf

Targeting Abasic Sites and Single Base Bulges in DNA with

Metalloinsertors

Brian M. Zeglis

Jennifer A. Boland

, and

Jacqueline K. Barton

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

California 91125

Maintaining the fidelity of the genome is critical to the cell, and DNA lesions threaten genetic

integrity.

Our laboratory has explored the design and applications of metalloinsertors that

target single base mismatches, a potential source of mutations in DNA.

–

The metal

complexes contain sterically expansive ligands that are too wide to fit into well-matched B-

form DNA and instead bind preferentially to thermodynamically destabilized mismatched

sites. These complexes, notably Rh(bpy)

(chrysi)

(chrysi = chrysene-5,6-quinone diimine)

(Figure 1), not only selectively bind to mismatched sites but also cleave the DNA backbone

upon photo-activation. Recent structural studies show that this mismatch binding occurs not

by intercalation, where the metal complex binds from the major groove, increasing the base

pair rise through stacking within the helix, but rather by insertion, where the bulky ligand of

the complex binds from the minor groove without increasing the base rise and ejects the

mismatched base pairs into the major groove.

This insertion mode clearly reconciles the

relationship between mismatch destabilization and site recognition: the more destabilized the

mismatch, the easier the extrusion of the mismatched bases.

The relationship between site destabilization and Rh(bpy)

-(chrysi)

affinity has led us to

investigate metalloinsertor recognition of two other common DNA defects: abasic sites and

single base bulges. Abasic sites result from cleavage of the glycosidic bond and can arise

spontaneously, as a result of exogenous carcinogens, or as an intermediate in repair.

Spectroscopic studies have shown abasic sites to destabilize the duplex by 3–11 kcal/mol.

Single base bulges, structurally related to abasic sites, are formed by replication errors. Bulged

sites are more stable than abasic sites, with destabilization values of

≤

4 kcal/mol.

Left

unrepaired, both structures can lead to cancer.

–

There are, however, few reports of targeting abasic sites and bulges in DNA and none with

high specificity. Higher reactivity of intercalators bearing nucleophilic amines with the abasic

aldehyde intermediate has been reported, but neither specific binding nor strand cleavage at

an abasic site is found.

–

Bulky intercalators have also been seen to react at multiple base

bulges or hairpin loops, but none bind to single base bulges and none with high specificity.

–

Such specific targeting of small deformations in DNA is important both fundamentally

in considering how these lesions may be detected within the cell and practically in the design

of diagnostics for these lesions. Here we report that our parent metalloinsertor, Rh

(bpy)

(chrysi)

, specifically binds and, with photoactivation, cleaves abasic sites and single

base bulges within duplex DNA.

E-mail: jkbarton@caltech.edu.

Supporting Information Available: Photocleavage titrations, gel analysis, and mass spectral data. This material is available free of charge

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

NIH Public Access

Author Manuscript

J Am Chem Soc

. Author manuscript; available in PMC 2009 October 9.

Published in final edited form as:

J Am Chem Soc

. 2008 June 18; 130(24): 7530–7531. doi:10.1021/ja801479y.

NIH-PA Author Manuscript

A series of oligonucleotides were synthesized to probe recognition of abasic sites and single

base bulges by Rh(bpy)

(chrysi)

(Figure 1).

Six duplexes, identical except for a central site,

were employed: well-matched (M), mismatched (MM), abasic site with unpaired cytosine (AB-

C), abasic site with unpaired guanine (AB-G), cytosine single base bulge (B-C), and guanine

single base bulge (B-G). Given the instability of the natural hemiacetal abasic site, the

tetrahydrofuranyl analogue was employed for the abasic strands.

Examination of the helix

melting points,

, of the DNA assemblies reveals that the abasic site and bulged base

significantly destabilize the duplex, in fact to a degree comparable to the CC mismatch (Figure

1).

Site-specific targeting by Rh(bpy)

(chrysi)

was examined in DNA photocleavage

experiments. As illustrated in Figure 2, Rh(bpy)

(chrysi)

specifically binds and, with

irradiation, cleaves not only mismatches but also abasic sites and single base bulges. In all

cases, cleavage neighbors the unpaired base and is evident only on one strand. The intensities

of the photocleavage bands for the abasic sites are comparable to that at the mismatch site;

photocleavage at the bulge sites is significantly diminished. This reduced intensity results both

from a lower binding affinity and from lower photocleavage efficiency; in fact, the poor

cleavage efficiency permits the determination of only an approximate affinity. For the abasic

sites, photocleavage titrations yield site-specific binding affinities of

∼

2 × 10

−

, values

comparable to that for the CC mismatch (see Figure 1). As expected for binding through

metalloinsertion, affinity correlates with the local destabilization.

In addition, the DNA cleavage products appear to mirror those seen with duplexes containing

the mismatch (Figure 2 and Supporting Information), consistent with an analogous binding

mode. Assayed by gel mobility, for each unpaired DNA assembly, we find a mixture of

cleavage products consistent with reaction to the 3

′

-side of the unpaired base to form 15-mers

containing a 3

′

-terminal phosphate along with 15-mers containing partially degraded 3

′

phosphoesters; heating or overnight incubation yields a tighter band that results from

decomposition of the mixture to the 3

′

-phosphate. Mass spectrometry has earlier been used to

identify products of Rh photocleavage of mismatched DNA.

Here, mass spectrometry results

for an abasic assembly yield a product distribution pattern analogous to that of mismatched

DNA. These products are consistent with H1

′

abstraction by the photoactivated metal complex

from the minor groove side, as expected with metalloinsertion.

For the bulged DNA

assemblies, gel analysis reveals bands of mobility similar to that created by mismatch cleavage,

indicative of reaction to the 3

′

-side of the bulged base.

Mass spectral data for the bulged

assemblies were inconclusive, however, owing to the low product yield.

The high site specificity in targeting abasic sites and bulged bases by Rh(bpy)

(chrysi)

coupled with the correspondence in affinities and cleavage products to mismatch targeting all

point to an analogous binding interaction of the metal complex with these structures versus

mismatched DNA. Figure 3 illustrates our model for Rh binding to an abasic site based upon

the crystal structure of Rh(bpy)

(chrysi)

bound to a CA mismatch.

In this model, the metal

complex inserts from the minor groove, displacing the unpaired base. The binding affinities

for mismatched DNA and an abasic site are equivalent, as are the

's, reflecting the

thermodynamic destabilization at the unpaired sites, the basis for ejecting the unpaired base.

The shared asymmetric cleavage and resultant products furthermore support a similar

orientation for the complex within the binding sites, with the Rh bound from the minor groove,

positioning the activated ligand for H1

′

abstraction. For the bulged DNA, the metal complex

is likely similarly positioned, but the binding pocket must differ, accounting for the poor

photocleavage efficiency. Nonetheless, for the bulged assemblies, based upon the cleavage

strand asymmetry and estimated binding affinities, a generally comparable binding mode and

orientation are likely.

Zeglis et al.

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J Am Chem Soc

. Author manuscript; available in PMC 2009 October 9.

NIH-PA Author Manuscript

These results establish that Rh(bpy)

(chrysi)

, a sterically bulky metalloinsertor, binds and

photocleaves abasic sites and single base bulges within duplex DNA with high affinity and

selectivity. The work thus expands the family of unpaired DNA structures targeted by Rh

(bpy)

(chrysi)

. Moreover, these data highlight the generality of site-specific binding to

destabilized structures within a DNA duplex through metalloinsertion.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We are grateful to the NIH (GM33309) for their financial support, the SURF program for a summer internship for

J.A.B, and the NSF for a graduate fellowship to B.M.Z.

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Figure 1.

-Rh(bpy)

(chrysi)

(left) along with sequences, melting temperatures (

), and binding

affinities (

) for the duplexes.

's were determined for 1

M DNA in buffer (50 mM NaCl,

10 mM NaPi, pH 7.1). Photocleavage titrations of DNA (1

M) in buffer and variable Rh

(bpy)

(chrysi)

(0–10

M) were employed to obtain site-specific binding constants (see

Supporting Information). “R” denotes tetrahydrofuranyl abasic site; “–” indicates no

nucleotide. Melting temperatures are accurate within 1 °C.

Zeglis et al.

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Figure 2.

Autoradiogram of a denaturing 20% polyacrylamide gel revealing photocleavage for Rh

(bpy)

(chrysi)

with the different DNA duplexes. 5

′

P-labeling is on the strand containing

′

-GAC. Conditions are duplex (1

M), Rh (3

M) in 50 mM NaCl, 10 mM NaPi, pH 7.1 for

30 min at ambient temperature followed by irradiation (0 or 30 min) with a solar simulator

(325–450 nm) and incubation at 60 °C (0 or 30 min). For matched DNA, lanes 1–3 contain

DNA with Rh without irradiation, DNA with Rh and 30 min irradiation, and DNA with Rh,

30 min irradiation, and 30 min at 60 °C, respectively. This set of three conditions is repeated

for each duplex: MM (lanes 4–6), AB-C (7–9), AB-G (10–12), B-C (13–15), and B-G (16–

18). The arrows mark the mispaired site.

Zeglis et al.

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Figure 3.

Model of Rh(bpy)

(chrysi)

inserted at an abasic site. The metalloinsertor is in red, the

extruded base in green, and the abasic deoxyribose in blue.

Zeglis et al.

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J Am Chem Soc

. Author manuscript; available in PMC 2009 October 9.

NIH-PA Author Manuscript