nihms122808.pdf

Insertion of a Bulky Rhodium Complex into a DNA Cytosine-

Cytosine Mismatch: An NMR Solution Study

Christine Cordier

†

Valerie C. Pierre

, and

Jacqueline K. Barton

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

California 91125, USA

Abstract

The bulky octahedral complex, Rh(bpy)

chrysi

(chrysi = 5, 6- chrysenequinone diimine), binds

single base mismatches in a DNA duplex with micromolar binding affinities and high selectivity.

Here we present an NMR solution study to characterize the binding mode of this bulky metal complex

with its target CC mismatch in the oligonucleotide duplex (5

′

-CGGA

TCCG-3

′

)

. Both NOESY

and COSY studies indicate that Rh(bpy)

chrysi

inserts deeply in the DNA at the mismatch site via

the minor groove and with ejection of both destabilized cytosines into the opposite major groove.

The insertion only minimally distorts the conformation of the oligonucleotide local to the binding

site. Both flanking, well-matched base pairs remain tightly hydrogen-bonded to each other, and 2D

DQF-COSY experiments indicate that all sugars maintain their original C2

′

endo

conformation.

Remarkably,

P NMR reveals that opening of the phosphate angles from a B

to a B

conformation

is sufficient for insertion of the bulky metal complex. These results corroborate those obtained

crystallographically and, importantly, provide structural evidence for this specific insertion mode in

solution.

INTRODUCTION

Octahedral metal complexes containing an extended bidentate aromatic ligand have been

designed to target single base mismatches in duplex DNA.

–

Rh(bpy)

chrysi

(chrysi = 5,6-

chrysenequinone diimine, Figure 1), is a sterically bulky DNA intercalator that binds

specifically in the destabilized regions near DNA base mismatches and, upon photoactivation,

cleaves the DNA backbone. The complex is both a general and remarkably specific mismatch

recognition agent.

Specific DNA cleavage is observed at over 80% of mismatch sites

irrespective of the sequence context around the mispaired bases. Furthermore, the complex

binds and with photoactivation cleaves at a single base mismatch in a 2725 base pair linearized

plasmid heteroduplex. This mismatch-specific targeting is based upon the thermodynamic

destabilization associated with the base mispair. The metal complex contains a bulky aromatic

bidentate ligand that is difficult to stack within well-matched DNA, but the extended complex

can insert more easily within the DNA duplex at the destabilized mismatched site.

*to whom correspondence should be addressed at jkbarton@caltech.edu.

†

Current address: ITODYS, Universite Denis Diderot, Paris VII, UMR CNRS 7086, 1 rue Guy de la Brosse, 75005 Paris, France.

Current address: Department of Chemistry, University of Minnesota, 207 Pleasant St., SE, Minneapolis, MN 55455, USA.

Supporting Information Available.

Table of nOe contacts of the free and Rh-bound oligonucleotide, chemical shifts of the chrysi ligand

in the presence and absence of DNA, list of nOe contacts observed between the chrysi ligand and the DNA, titration of

-Rh(bpy-

)

chrysi

to DNA containing CA versus CC mismatches, melting curves of the oligonucleotides with and without the metalloinsertor,

mass spectra of photocleavage experiments, COSY and NOESY spectra of free

-Rh(bpy-

)

chrysi

, 2D DQF-COSY and NOESY

subspectra of the free oligonucleotide, NOESY subspectra of the exchangeable protons of the DNA bound to the metalloinsertor. This

information 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 September 22.

Published in final edited form as:

J Am Chem Soc

. 2007 October 10; 129(40): 12287–12295. doi:10.1021/ja0739436.

NIH-PA Author Manuscript

The uniquely high specificity in targeting mismatches has led to many applications for these

bulky metal complexes. The mismatch-targeting agents have been fruitful in developing new

methods for the discovery of single nucleotide polymorphisms.

Pooling and annealing

together DNA samples from a test population generates mismatches at the polymorphic sites,

and these can be marked by photocleavage with the mismatch-specific complex. Additionally,

since there is an association between deficiencies in mismatch repair and cancerous

transformation,

–

these complexes may provide a route to novel diagnostics for cancer.

Measurements of the abundance of mismatches provides an early report on deficiencies in

mismatch repair.

Towards that end, we have developed luminescent analogues as probes for

mismatches.

Bifunctional complexes have also been designed to target alkylators and

platinating agents to mismatched sites.

Moreover, we have found that these bulky

rhodium intercalators can differentially inhibit cellular proliferation in mismatch repair-

deficient cells compared with cells that are mismatch repair-proficient.

Significantly, then,

targeting DNA mismatches may provide a new cell-selective strategy for chemotherapeutic

design.

–

Given the interest in applying these complexes for a range of biological applications, a

structural understanding of their interactions with mismatched DNA becomes critically

important. The crystal structure of

-Rh(bpy)

chrysi

bound to a CA mismatch within a DNA

oligonucleotide was recently determined.

The structure revealed that the metal complex

recognizes the thermodynamically destabilized site via a novel binding mode:

insertion into

the double helix with ejection of both mismatched bases

. Importantly, this insertion mode

differs from the well characterized intercalation mode

–

in that the DNA does not unwind

to enable introduction of a new smaller ligand in its base stack, but replaces and ejects the

faulty mispair using the extended ligand. Indeed, given the width of the chrysi ligand (11.3 Å),

wider than the span of a base pair (10.8 Å), insertion of the metal complex to a destabilized

site is favored over intercalation in stable, matched DNA. The crystal structure thus

corroborates the direct correlation observed between the binding affinity of the metalloinsertor

for a given mismatch and the amount by which that mismatch destabilizes DNA.

The crystal structure revealed many distinctive aspects of this binding mode, (i) insertion into

the duplex stack without increasing the base pair rise, (ii) ejection of both mismatched bases,

and (iii) binding to the DNA from the minor groove side. This mode is clearly distinguished

from the structurally characterized intercalation of metal complexes which has been found to

give a doubling of base pair rise at the intercalation site, no base pair opening, and binding

from the major groove side.

–

In fact, in the crystal structure of the mismatched DNA, an

additional Rh complex is bound at the center of the helix through the characteristic intercalative

mode.

We attributed this extra complex to be bound as a result of crystal packing forces.

Given this observation, it is important also to obtain structural information regarding these

binding interactions in solution. Are the bases fully ejected in solution as in the solid state,

where additional stacking interactions help to stabilize the ejected mismatched bases? Does

the interaction arise from the minor or major groove side? Are there significant perturbations

in the sugar-phosphate backbone and are they local to the site or extended over a portion of

the duplex? Moreover, it is most important to ascertain not only whether the observations made

for the crystal hold as well in solution, but also that the observations can be extended to other

mismatches and other duplex sites. Thus we describe here, using

H and

P-NMR, a solution

study of the insertion of

-Rh(bpy)

chrysi

into its target CC mismatch within an

oligonucleotide DNA duplex.

Cordier et al.

Page 2

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

RESULTS AND DISCUSSION

Choice of oligonucleotide

Rh(bpy)

chrysi

binds single base mismatches with affinities of 10

–10

−

depending on

the thermodynamic destabilization associated with the mismatch. Our initial study thus

concentrated on characterizing the insertion of the bulky metal complex into the sites to which

it binds most tightly, the most destabilizing CC and CA mismatches.

Several oligonucleotides

containing either 1 or 2 mismatches and varying both in length and % GC content were thus

evaluated. In most cases, titration of 1 equivalent of Rh complex/mismatch results in broad

NMR spectra, characteristic of an intermediate exchange process, and not useful for structure

determination (Figure S1, supporting information). Although not sufficient for a detailed

structural analysis, sharper spectra are obtained with a palindromic 9-mer containing a central

CC mismatch: 5

′

-C

-3

′

. It was therefore this sequence that was used for

this NMR study. It should be noted that changing the ionic strength or the pH between 6 and

8 of the DNA/Rh complex solution does not sharpen the spectra, nor does addition of spermine.

Furthermore, since the melting point of the oligonucleotide in the absence of the metalloinsertor

is barely 18 °C, all NMR experiments, both in the presence and absence of Rh

(bpy)

chrysi

, were thus performed at or below 10 °C so as to ensure that the DNA maintains

a fully duplex form.

Binding of

-Rh(bpy-d

)

chrysi

to the CC mismatch

Previous studies with octahedral tris- chelate complexes demonstrated their binding preference

for DNA of matching chirality.

In each case, it is the

enantiomer that preferentially

intercalates into right-handed B-DNA.

–

In the case of metalloinsertors, this chiral

discrimination is even more dramatic. Only the

enantiomer of Rh(bpy)

chrysi

effectively

binds DNA, and minimal interaction is observed with the

enantiomer.

For the purpose

of this study, the

and

enantiomers were thus separated by chiral chromatography using

antimonyl tartrate as an eluent and only the

enantiomer was titrated with the DNA.

Furthermore, in order to simplify the aromatic region of the NMR spectra and avoid overlap

between the resonances of the protons on the ancillary ligands and those of the bases, the

metalloinsertor was synthesized with deuterated bipyridines.

As commonly observed with complexes that stack within the duplex, insertion of

-Rh(bpy-

)

chrysi

in the oligonucleotide containing a CC mismatch significantly stabilizes the DNA

duplex by 19 °C (Figure S2, supporting information), consistent with insertion of the chrysi

ligand inside the DNA base stack. Furthermore, photocleavage experiments on the 9-mer

followed by MALDI-TOF mass spectrometry indicate a single cleaving point neighboring

, consistent with insertion of the Rh complex at the CC mismatch (Figure S3, supporting

information).

Thus, independent of the NMR results, photocleavage experiments and melting

temperature studies indicate that

-Rh(bpy-

)

chrysi

does insert in the oligonucleotide

selectively at the CC mismatch.

Assignments and structure of the mismatched DNA without metal Complex

The free, unbound oligonucleotide 5

′

-CGGACTCCG-3

′

was characterized by NOESY,

HOHAHA, DQF-COSY and

P NMR spectroscopy. Concomitant assignment of the NOESY,

HOHAHA, and DQF-COSY spectra confirmed that the mismatch-containing oligonucleotide

is in a duplex form and adopts the regular B conformation.

The two self-complementary

strands are related by C2 symmetry. All bases maintain the standard

anti

conformation. The

pattern of the H

′

-H

′

DQF-COSY crosspeaks, together with the absence of H

′

-H

′′

correlations and the relative intensities of the crosspeaks in the NOESY spectrum all confirm

that every sugar pucker is predominantly in the C

′

endo

conformation (Figure S4, supporting

information). Note that the H

′

and H

′′

chemical shifts are inverted for the terminal G9 and

Cordier et al.

Page 3

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

the mismatch compared to the other nucleotides of the sequence. Furthermore, the

P 1D NMR

spectrum indicates that the CC mismatch does not significantly distort the backbone of the

DNA, as all phosphodiester junctions are in the canonical B

conformation (

vide infra

Importantly, there is no break in nOe connectivity along the strand (Figure 2). All base pairs

are therefore canonically stacked, including the mismatched cytosines. Insertion of the bulky

metal complex into the mismatched site is thus not a matter of finding a “hole” in the DNA

base stack, but of breaking apart weakly paired bases and ejecting them out of the double helix.

The NOESY spectrum recorded in H

O at 4 °C enables the assignment of the exchangeable

imino protons of the bases (Figure S5, supporting information). All matched bases are paired

in the normal Watson-Crick mode. We were unable to identify the amino protons of the CC

mismatch, however. This observation was also previously reported for a CC mismatch studied

under slightly acidic conditions.

–

Since the two cytosines adopt the

anti

conformation,

they may be paired according to a Wobble-type of conformation in which a single amino

hydrogen bonds the two cytosines. Since this hydrogen bond would be in rapid equilibrium

between two conformations, its exchange with bulk solvent is facilitated and its signal is

thereby reduced.

NMR characterization of the insertion

Titration of the metalloinsertor into the oligonucleotide results in significant line broadening

(Figure S1, supporting information). Nonetheless, most protons can be assigned by a

combination of NOESY, HOHAHA, and 2D DQF COSY starting from the resonance of the

′

. Significantly, the 1D

H NMR spectra for the titration clearly indicate that the aromatic

protons of the chrysi ligand shift significantly upfield upon addition of the DNA, consistent

with its insertion and stacking inside the DNA base stack.

Intraduplex

H-correlations—

Notably, both the NOESY and the HOHAHA spectra

recorded in D

O clearly indicate the loss of the C

symmetry in the central part of the

oligonucleotide upon insertion of the rhodium complex (Figures 2 and 3). For instance, two

thymine H

/Me correlations and five cytosine H

correlations are observed in the presence

-Rh(bpy-

)

chrysi

, as opposed to only one and four respectively for free DNA (Figure

3). Two strands, labeled a and b, are thus clearly distinguished in the central part of the

oligonucleotide comprising the A

steps. Accordingly, in the NOESY spectra, two

sequential NOESY walks, labeled a and b, were built along the central part of the DNA (Figure

2b). These NOESY data confirm that the double helix maintains its original B-conformation

upon insertion of the chrysi ligand despite its extended width.

Importantly, no nOes are observed in either strand between T

Me and C

. These nOes are,

however, clearly present in the free oligonucleotide. Furthermore, the nOes observed between

and C

clearly indicate that, in both strands, the mismatched cytosines are still close to

their neighboring adenosines. Together, these observations are consistent with ejection of the

mismatched cytosines from the double helix asymmetrically in such a way that they remain

closer to the purines than the pyrimidines. Furthermore, the crosspeaks assigned to the C

from

strand b are broader than those from strand a. This suggests that one of the ejected cytosines

is more flexible and less constrained than the other.

No anomalous intra nOe crosspeaks for the bases H

and H

are detected suggesting that, even

after insertion, all bases maintain their

anti

conformation. Furthermore, the patterns of the 2D

DQF- COSY crosspeaks, although more difficult to observe in the Rh-bound DNA than in the

free oligonucleotide, suggest that all sugars, including the ejected cytosines, maintain their

′

endo

puckering (Figure 4). This is further supported by the absence of correlations between

′

and H

′′

Cordier et al.

Page 4

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Again, recording NMR spectra in H

O at 4 °C enables the assignment of the exchangeable

imino protons (Figures S6 and S7, supporting information). The T

a and T

b imino protons

were assigned according to their dipolar correlations with T

aMe and T

bMe respectively, as

well as with A

a and A

b to which they are paired. These T

imino protons markedly shift

upfield upon insertion of the chrysi ligand. Again, no correlations are observed between the

imino protons of the mismatched cytosines. Yet, in this instance, the lack of contact between

a and C

b is more likely due to their ejection from the DNA base stack than to any Wobble-

type pairing between them.

P-NMR results—

The more flexible part of the DNA, namely the phosphodiester backbone,

undergoes significant distorsion upon insertion. Addition of

-Rh(bpy-

)

chrysi

significantly changes the 1D

P NMR spectrum of the oligonucleotide: at least one peak shifts

significantly downfield from the others (Figure 5). Gorenstein and coworkers previously

reported that the torsion angle difference (

) can be directly calculated from the chemical

shifts of the

P NMR spectrum.

The resulting (

) values directly indicates whether the

phosphodiester backbone is in the B

or B

conformation. In the case of the free

oligonucleotide, all phosphodiester junctions are in the normal B

conformation. However, in

the Rh-bound DNA, while most of the phosphates have chemical shifts that clearly indicate

torsion angles for the B

conformation, one of the phosphates, that which is shifted downfield,

has shifts that reflect adoption of the more open B

conformation.

H-correlations between the DNA and metal Complex—

The

H NMR signals of the

free

-Rh(bpy-

)

chrysi

were first assigned by the combination of its NOESY and COSY

spectra (Figure S8, supporting information). As a reminder, since the ancillary bipyridines are

deuterated, they remain silent during the

H NMR experiments. The two exchangeable

and

protons give rise to an additional signal in H

O (4 °C) at

= 8.70 ppm.

Intermolecular correlations between the chrysi ligand and the central part of the oligonucleotide

are also observed and give insights into the insertion binding mode. About twenty specific

contacts are detected between the chrysi ligand and the oligonucleotide at the site of insertion

(Figure 6 and Table S4, supporting information), principally with the ejected C

and T

. The

chrysi protons advantageously shift upfield upon insertion in a frequency window without

strong overlap, such that many contacts with sugar protons can be assigned. However, the

′

(

= 7.97 ppm) which would have been a valuable indicator to probe the minor groove

occupancy by the metalloinsertor, overlaps significantly with some chrysi protons (

= 7.93

ppm), such that the corresponding intermolecular contacts are masked by the strong intra-chrysi

correlations.

The intra-DNA nOes observed between A

and C

′

of strand a are consistent with ejection

of the mismatched C

a in the major groove (Figure 2). The homologous contact with C

b was

however not detected even at lower signal to noise ratio. Nonetheless, the position of the second

mismatched cytosine, C

b, in the major groove could be ascertained by intermolecular contacts

with the chrysi ligand (Figure 7). Indeed, the correlation observed between the sugar protons

of C

b located in the minor groove (H

′

and to a lesser extend, H

′

) and the aromatic chrysi

proton clearly also position C

b in the major groove. Additional nOe correlations between the

chrysi ligand and the H

′

, H

′′

and H

′

of T

of both strands as well as the lack of nOe between

Me and C

further support insertion of the rhodium complex via the minor groove and

ejection of both mismatched cytosines in the opposite major groove. The strong dipolar

crosspeaks observed between the chrysi ligand and the T

Me confirm the deep insertion of Rh

(bpy-

)

chrysi

Cordier et al.

Page 5

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Changes in chemical shift associated with binding

Changes in chemical shifts also give valuable information on the binding of the bulky metal

complex to DNA. Upon insertion in the oligonucleotide, most of the protons of the chrysi

ligand shift upfield by as much as

−

2.05 ppm (Figure 8), consistent with insertion and

stacking of the bulky ligand inside the DNA base stack.

–

Notably, one of the chrysi

protons,

, shifts slightly downfield, while its neighbor,

, shifts significantly upfield. These

two unusual shifts may be explained by insertion of the rhodium complex from the minor

groove. Indeed, this minor groove orientation places

directly below the carbonyl of T

which may perturb the ring current, while

in the

ortho

positions is directly placed under the

ring current and thus undergoes a significant upfield shift.

Just as the metalloinsertor undergoes significant chemical shifts upon insertion, so does the

DNA (Figure 9). For clarification, only those protons which shift more than 0.1 ppm are

considered; also the H

′

and H

′′

are not considered since they strongly overlap with each other.

Nonetheless, as is evident from the bar graph, the central part of the oligonucleotide marking

the site of insertion, namely A

, undergoes a significantly greater shift, reflecting a

greater change in magnetic surrounding, than does the rest of the DNA. Importantly, the protons

of both mismatched cytosines (C

a and C

b) shift downfield, consistent with their ejection

from the base stack. The exception of C

′

could be explained by the fact that the H

′

and

′′

of C

are inverted in the free oligonucleotide. Similarly, most protons of the flanking

·T

base pairs are shifted upfield due to their efficient

-stacking with the inserted chrysi

ligand. Significantly, the H

′

of the flanking A

·T

base pairs shift upfield, consistent with

insertion of

-Rh(bpy-

)

chrysi

via the minor groove (

Δδ

−

0.24 ppm,

−

0.31 ppm, and

−

0.37 ppm for A

, T

a and T

b respectively).

The exchangeable imino protons of the flanking T

also undergo significant upfield shifting

(

Δδ

−

1.61 ppm) and this dramatic shift is another strong indicator of the deep insertion of

the chrysi ligand. This thymine is still tightly hydrogen bonded to its adenine; no changes are

observed for the base pairs flanking the extended chrysi ligand. Indeed, analysis of the amino

protons of the adenosines indicate that the chemical shift differences between the free and

bonded amino,

Δδ

amino

are 1.46 ppm and and 1.12 ppm for A

of strands a and b respectively.

This

Δδ

amino

is a direct indicator of the strength of the base pairing and is comparable to that

of the triply hydrogen-bonded C

, C

and C

(

Δδ

amino

= 1.43 ppm, 1.52 ppm and 1.53 ppm

respectively).

Notably, homologous protons in strand a and b of the oligonucleotide are perturbed differently,

resulting in the break in the C

symmetry at the site of insertion. Strand b of the DNA undergoes

greater changes in chemical shift than does strand a, suggesting that the wider part of the chrysi

ligand (corresponding to the protons

7–10

) is pointed toward strand b.

Insertion mode

Figure 10 displays the basic characteristics associated with insertion of the bulky metal

complex into the mismatched DNA that are evident from the NMR study. Consistent with the

chemical shift changes seen both for the metal complex and for the DNA as well as the NOESY

data, the metal complex is clearly bound at the central mismatched site by insertion and stacking

of the chrysi ligand. This leaves sufficient room for the ancillary ligands of the

-isomer but

not the

-isomer to insert into the duplex, if there is no increase in base pair rise at the insertion

site. Based upon the NOESY data, the metal complex binds from the minor groove side and

inserts deeply into the duplex so that the chrysi protons can interact with major groove protons.

The cytosines are ejected out into the major groove, in an asymmetric, likely more flexible,

fashion; no stacking or hydrogen bonding by the mismatched cytosines occur. The insertion

Cordier et al.

Page 6

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

mode is, furthermore, local to the mismatched site, and the remainder of the duplex remains

in a hydrogen-bonded and stacked B-conformation.

Comparison with the crystal structure

Taken together, the NMR solution study and the solid state structure obtained from single

crystal X-ray diffraction

give a clear picture of the novel insertion binding mode of the bulky

metal complex with a single base mismatch. Since the crystal structure and the NMR studies

were performed on different oligonucleotides containing different mismatches (CA and CC

mismatches respectively) and the results are seen to be mutually consistent, it appears that the

features seen here reflect insertion of the complex inside

any

thermodynamically destabilized

mismatched duplex.

Significantly, insertion differs from the previously well characterized intercalation

–

in that the DNA does not unwind to enable a ligand to enter the base stack, but rather the

incoming ligand simply ejects both bases of the destabilized or weakly bonded pair. The present

NMR study confirms that minimal perturbation of the DNA is required for insertion of the

bulky ligand and base ejection. Both studies indicate that all sugars maintain their C

′

endo

puckering and all bases maintain their

anti

conformation, including the ejected ones. The

oligonucleotide enables insertion of the chrysi ligand by opening its phosphodiester junctions

from a B

to a more open B

conformation. The present NMR study also clearly confirms that,

even in solution, the base pairs flanking the chrysi ligand remain paired, and this despite the

extended length of chrysi, 0.5 Å wider than the span of a base pair.

Thus

-Rh(bpy-

)

chrysi

can discriminate single base mismatches effectively. The present

NMR solution study and the crystal structure

illustrate this ejection of destabilized bases by

the bulky rhodium complex without perturbing well-paired neighboring sites. Both studies

demonstrate that there is sufficiently deep insertion of the chrysi ligand so that it protrudes

through the base stack and into the opposite major groove. Both studies also reveal the break

in symmetry upon insertion of the chiral metal complex. Importantly, this results in two ejected

bases of different nature. Indeed, both studies indicate that one of the ejected bases is

significantly more flexible than the other. In the crystal structure, the more flexible base (in

that case an adenosine) is able to fold back into the minor groove where it is

-stacked with

both an ejected purine and an ancillary bpy ligand of a rhodium complex inserted in a nearby

crystallographically related oligonucleotide; such an interwoven conformation is unlikely in

solution. Indeed, the present study illustrates that in solution, although more flexible, the second

mismatched base is still positioned in the major groove.

Remarkably, this difference in flexibility between the two ejected bases is in accordance with

the photoactivated cleavage experiments performed with this class of compounds.

In all

cases, only one strand of the DNA is cleaved upon photoactivation of the inserted rhodium

complex. This corresponds, both in solution and in the solid state, to the strand with the more

rigid ejected base. Furthermore, both studies place the sugar protons of the base flanking the

mismatch closer to the chrysi ligand than the sugar of the ejected base, thereby explaining why

photoactivation of the inserted rhodium complex cleaves the base one away from the mismatch

and not at the mismatch itself.

It should be noted that there is, however, one clear difference between the present solution

study and the crystal structure. In the crystal structure, an additional Rh complex is bound by

intercalation from the major groove side at the central 5

′

AT-3

′

step.

Here, while there is

significiant broadening of all the resonances in the bound form, there is no direct indication of

any specific perturbation of the well-matched DNA sites, only binding at the mismatched site.

This lack of evidence for binding at a well-matched site in solution is consistent, in the crystal,

with binding at the matched site being a result of crystal packing forces. Also consistent with

Cordier et al.

Page 7

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

this difference between solid state and solution, we had observed photoactivated cleavage at

the matched site as well as the mismatched sites in the crystal but only at the mismatched site

in solution.

CONCLUSION

The NMR solution study of

-Rh(bpy-

)

chrysi

bound to its target CC mismatch in DNA

reveals the key features associated with insertion into a mismatched DNA site. Bulky

metalloinsertors can target destabilized single-base mismatches, replacing the weakly bonded

bases and ejecting them out of the DNA base stack. The insertor enters the DNA via the minor

groove, thereby pushing the ejected bases into the opposite major groove. This observation

corroborates the direct correlation between the binding affinity of

-Rh(bpy-

)

chrysi

for

mismatches and the extent to which these mismatches destabilize DNA.

–

The C

symmetry

of the palindromic oligonucleotide is broken upon insertion of the rhodium complex resulting

in two distinct strands. Consequently, the two ejected bases are no longer equivalent: one of

the two mismatched cytosines is significantly more flexible than the other. This result is in

accordance with previous photoactivated cleavage experiments. Notably, insertion of the bulky

metalloinsertor also only minimally and locally distorts the structure of the DNA duplex. All

bases maintain their

anti

conformation and the sugars maintain their regular C

′

endo

puckering, including the ejected ones; insertion of the bulky ligand is accomodated only by a

small change in the phosphodiester junction.

EXPERIMENTAL SECTION

Materials

Unless otherwise noted, starting materials were obtained from commercial suppliers and used

without further purification. Phosphoramidites, reagents and solid supports for DNA synthesis

were obtained from Glen Research. Trimethylphosphate (TMP) and bipyridine-

were

obtained from Aldrich. RhCl

was obtained from Pressure Chemicals. D

O (99.96 %) and 2,2-

dimethyl-2-silanepentane-5-sulfonate sodium (1% DSS-

in 99.99% D

O) were obtained

from Cambridge Isotopes Labs and Itotec respectively. Water was deionized and further

purified by a Millipore cartridge system (resistivity 18 × 10

Rh(bpy-

)

chrysi

was synthesized according to literature procedure using bpy-

The

two enantiomers were separated as previously described using chromatography with the chiral

eluant, antimonyl tartrate.

Only the

enantiomer was titrated into the DNA. The self-

complementary oligonucleotide, 5

′

-CGGACTCCG-3

′

was synthesized on an Applied

Biosystems 394 automatic DNA synthesizer and purified according to standard protocols.

The DNA concentrations of all samples were determined by UV-visible spectroscopy at 70 °

C on a Beckman DU 7400 diode array spectrophotometer equipped with a Peltier heating

sample holder. An extinction coefficient for the single stranded oligonucleotide of

260

ssDNA

= 88,200 M

−

was used. The melting profiles were similarly measured with a heating and

cooling rate of 0.5 °C/min between 5 °C and 50 °C.

Photocleavage experiments

Photoactivated cleavage was performed as previously described

using the following

concentrations: 7.5

M dsDNA, 7.5

-Rh(bpy-

)

chrysi

(1 equivalent/mismatch), 10

mM Na phosphate, 50 mM NaCl, pH 7.0. Irradiation was carried out at 313 nm using a 1000

W Oriel Hg/Xe arc lamp with a monochromator fitted with a 300 nm cutoff filter and IR filter.

MALDI-TOF mass spectra were recorded on a PerSeptive Biosystem Voyager-DE Pro.

Cordier et al.

Page 8

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

NMR sample preparations

The NMR sample of the free oligonucleotide was prepared by dissolving the lyophilized 9-

mer in 500

L of 50 mM Na phosphate buffered at pH = 6.10 containing 20 mM NaCl.

The

final concentration of duplex DNA was 2.33 mM. For spectra measured in D

O, the solvent

was lyophilized from the aqueous buffer, then twice redissolved in 99.9 % D

O and lyophilized,

and finally redissolved in 500

L of 99.96 % D

O. A drop of 1 % DSS-d

in D

O was added.

The DSS- d

methyl signal was used as internal reference (

= 0.000 ppm). For investigation

of the exchangeable protons, the D

O sample was lyophilized and redissolved in H

O/D

90/10.

The NMR sample of Rh-bound DNA was similarly prepared with a final concentration of 1.62

mM duplex DNA, 1.62 mM

-Rh(bpy-

)

chrysi

(1 equivalent/mismatch), 50 mM Na

phosphate buffered at pH = 6.10, 20 mM NaCl. Likewise, the NMR sample of the free

metalloinsertor contained 1.62 mM

-Rh(bpy-

)

chrysi

, 50 mM Na phosphate buffered at

pH = 6.10, 20 mM NaCl.

NMR Measurements

H NMR spectra were collected on a Varian Unity-Plus 600 spectrometer equipped with a

variable temperature unit and pulse-field gradients in three dimensions.

P NMR spectra were

recorded at 242 MHz on a Varian 300 MHz instrument.

H and

P NMR spectra were recorded

using a dual (

C) and a quadra (

P) probes respectively. NMR data were

processed using the MestReC software (version 4.8.6.0).

The non-exchangeable (D

O) and exchangeable (H

O/D

O: 90/10) proton spectra were

recorded at 10 °C and 4 °C respectively. NOESY spectra in D

O of the free DNA and

-Rh

(bpy- d

)

chrysi

inserted in DNA were collected with mixing times of 150 and 300 ms

respectively (12 ppm sweep width, TPPI, 2048 complex points, 560 t1 blocks, 64 scans per t1

block, 2.5 s relaxation delay, water suppression by presaturation of the residual signal during

relaxation time and mixing time). WaterGATE NOESY spectra in H

O/D

O (90/10) were

recorded with a mixing time of 300 ms for the free DNA and

-Rh(bpy-d

)

chrysi

inserted

in DNA (20 ppm sweep width, TPPI, 4096 complex points,560 t1 blocks, 96 scans per t1 block,

2 s relaxation delay). HOHAHA experiments were collected with a mixing time of 110 ms so

as to optimize the coherence transfers from the H1

′

to H2

′

and H2

′′

protons

(12 ppm sweep

width, hyper-complex mode, TPPI, 2048 complex points, 380 t1 blocks, 64 scans per t1 block,

1.5 s relaxation delay, water suppression by presaturation of the residual signal). DQF-COSY

experiments

were collected with 512 and 680 t1 blocks for the free DNA and the Rh-bound

DNA respectively (12 ppm sweep width, TPPI, 4096 complex points, 64 scans per t1 block,

1.5 s relaxation delay, water suppression by presaturation of the residual signal). 1D

P NMR

experiments were recorded at 10 °C and referenced to an external standard of TMP (0.1 M in

= 0.000 ppm, 20 ppm sweep width, 4096 complex points, 250 and 800 scans, 1.0 s

relaxation delay).

For the free Rh(bpy-

)

chrysi

an additional set of COSY and NOESY experiments were

performed in D

O at 20 °C (COSY: 9 ppm sweep width, 2048 complex points, 512 t1 blocks,

16 scans per t

block, 1.0 s relaxation delay, water suppression by presaturation of the residual

signal; NOESY: 9 ppm sweep width, 2048 complex points, 640 t

blocks, 16 scans per t

block,

water suppression by presaturation of the residual signal during 1.0 s relaxation time and 0.8

s mixing time). Exchangeable protons of the Rh complex were assigned in H

O/D

O: 90/10

at 4 °C by 1D NMR (12 ppm sweep width, 32 K complex points, 16 scans, 1.0 s relaxation

delay).

Cordier et al.

Page 9

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We are grateful to the National Institutes of Health (GM33309) for their financial support. We also thank the Universite

D. Diderot (Paris 7) for sabbatical support to C. C. Additionally we thank P. K. Bhattacharya, K. Crowhurst, M.

Shahgholi, and S. Ross for helpful discussions.

References

1. Jackson BA, Barton JK. J Am Chem Soc 1997;119:12986–12987.

2. Jackson BA, Barton JK. Biochemistry 2000;39:6176–6182. [PubMed: 10821692]

3. Jackson BA, Alekseyev VY, Barton JK. Biochemistry 1999;38:4655–4662. [PubMed: 10200152]

4. Hart JR, Johnson MD, Barton JK. Proc Natl Acad Sci U S A 2004;101:14040–14044. [PubMed:

15383659]

5. Kunkel TA, Erie DA. Annu Rev Biochem 2005;74:681–710. [PubMed: 15952900]

6. Strauss BS. Mutation Research/Reviews in Mutation Research 1999;437:195–203.

7. Arzimanoglou II, Gilbert F, Barber HRK. Cancer 1998;82:1808–1820. [PubMed: 9587112]

8. Loeb LA, Loeb KR, Anderson JP. Proc Natl Acad Sci U S A 2003;100:776–781. [PubMed: 12552134]

9. Junicke H, Hart JR, Kisko J, Glebov O, Kirsch IR, Barton JK. Proc Natl Acad Sci U S A 2003;100:3737–

3742. [PubMed: 12610209]

10. Ruba E, Hart JR, Barton JK. Inorg Chem 2004;43:4570–4578. [PubMed: 15257584]

11. Zeglis BM, Barton JK. J Am Chem Soc 2006;128:5654–5655. [PubMed: 16637630]

12. Schatzschneider U, Barton JK. J Am Chem Soc 2004;126:8630–8631. [PubMed: 15250697]

13. Petitjean A, Barton JK. J Am Chem Soc 2004;126:14728–14729. [PubMed: 15535691]

14. Hart JR, Glebov O, Ernst R, Kirsch IR, Barton JK. Proc Natl Acad Sci U S A 2006;103:15359–15363.

[PubMed: 17030786]

15. Brunner J, Barton JK. Biochemistry 2006;45:12295–12302. [PubMed: 17014082]

16. Puckett CA, Barton JK. J Am Chem Soc 2007;123:46–47. [PubMed: 17199281]

17. Pierre VC, Kaiser JT, Barton JK. Proc Natl Acad Sci U S A 2007;104:429–434. [PubMed: 17194756]

18. Erkkila KE, Odom DT, Barton JK. Chem Rev 1999;99:2777– 2795. [PubMed: 11749500]

19. Sitlani A, Long EC, Pyle AM, Barton JK. J Am Chem Soc 1992;114:2303–2312.

20. Kielkopf CL, Erkkila KE, Hudson BP, Barton JK, Rees DC. Nat Struc Biol 2000;7:117–121.

21. Barton JK. Science 1986;233:727–734. [PubMed: 3016894]

22. David SS, Barton JK. J Am Chem Soc 1993;115:2984–2985.

23. Sitlani A, Dupureur CM, Barton JK. J Am Chem Soc 1993;115:12589–12595.

24. Hiort C, Lincoln P, Norden B. J Am Chem Soc 1993;115:3448–3454.

25. (a) Dupureur CM, Barton JK. Inorg Chem 1997;36:33. (b) Zeglis BM, Barton JK. Nature Prot

2007;2:357–371.

26. Brunner J, Barton JK. J Am Chem Soc 2006;128:6772–6773. [PubMed: 16719441]

27. Wuthrich, K. NMR of Proteins and Nucleic Acids. Wiley; New York: 1986. James, TL., editor.

Nuclear Magnetic Resonance and Nucleic Acids. Vol. 261. Academic Press, Inc; San Diego: 1995.

Methods in Enzymology.

28. Van de Ven FJ, Hilbers CW. Eur J Biochem 1988;178:1–38. [PubMed: 3060357]

29. Kouchakdjian M, Li BFL, Swann PF, Patel DJ. J Mol Biol 1988;202:139–155. [PubMed: 2845094]

30. Gray DM, Cui T, Ratliff RL. Nucl Acids Res 1984;12:7565–7580. [PubMed: 6493980]

31. Boulard Y, Cognet JAH, Fazakerley GV. J Mol Biol 1997;268:331–347. [PubMed: 9159474]

32. (a) Patel DJ, Shapiro L. J Biol Chem 1986;261:1230. [PubMed: 3003062] (b) Patel DJ, Shapiro L.

Biopolymers 1986;25:707. [PubMed: 3011140] (c) Aggarwal A, Islam SA, Kuroda R, Neidle S.

Biopolymers 1984;23:1025–1041. [PubMed: 6733246] (d) Weiner SJ, Kollman PA, Nguyen DT,

Cordier et al.

Page 10

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Case PA. J Comp Chem 1986;7:230. (e) Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C,

Alagana G, Piotete S, Weiner P. J Am Chem Soc 1984;106:765. (f) LaMar G, VanHecke G. Inorg

Chem 1970;9:1546. (g) Huang T, Brewer D. Can J Chem 1981;59:1689.

33. Gorenstein DG. Chem Rev 1994;94:1315–1338.The empirical relation of Gorenstein is (

–

= 254.5

+ 72.8

34. Franklin SJ, Barton JK. Biochemistry 1998;37:16093–16105. [PubMed: 9819202]

35. Dupureur CM, Barton JK. J Am Chem Soc 1994;116:10286–10287.

36. Collins JG, Shields TP, Barton JK. J Am Chem Soc 1994;116:9840–9846.

37. Hudson BP, Barton JK. J Am Chem Soc 1998;120:6877–6888.

38. Murner H, Jackson BA, Barton JK. Inorg Chem 1998;37:3007–3012.

39. pD = pH + 0.4 at 25 °C. The electrode (pH) was calibrated with standard buffered solutions. The pH

values indicated correspond to the measured and uncorrected value.

40. Cavanagh J, Chazin WJ, Rance MJ. J Magn Reson 1990;87:110–131.

41. Wijmenga, S.; Mooren, MW.; Hilbers, C. NMR of Macromolecules, a Practical Approach. Roberts,

GCK., editor. Oxford University Press; Oxford: 1993. p. 217-288.

42. Piantini U, Sorensen OW, Ernst RR. J Am Chem Soc 1982;104:6800–6801.

Cordier et al.

Page 11

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 1.

Chemical structure of

-Rh(bpy-

)

chrysi

with numbering scheme for the protons of the

chrysi ligand and sequence and numbering scheme for the oligonucleotide.

Cordier et al.

Page 12

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 2.

NOESY data for the free oligonucleotide and in the presence of metal complex. (

) (F2 × F1:

′

× aromatic) and (

) (F2 × F1: Me × aromatic) NOESY sub-spectra of the free DNA showing

the sequential walk of the nOes along the full strand (

). No break in the correlation network

confirms the intrahelical stacking of the two mismatched cytosines inside the double helix.

Experimental conditions: D

O, 10 °C, 300 ms mixing time. (

) NOESY sub-spectrum of

Rh(bpy-

)

chrysi

inserted in DNA and sequential walk of the nOes along the full strand.

(F2 × F1: aromatic × H

′

). The loss of the C

symmetry in the A

step of the DNA is

highlighted by two chains of connectivities displayed in dark blue (strand a) and light green

(strand b). The nOe between G

and C

(marked by *) is observed at lower signal to noise ratio.

Intramolecular nOe correlations between chrysi protons (

) is labeled

. Experimental

conditions: D

O, 10 °C, 300 ms mixing time. (

) TOCSY sub-spectrum of

-Rh(bpy-

)

chrysi

inserted in DNA. (F2 × F1: H2

′

-H2

′′

× H1

′

). Experimental conditions: D2O, 10 °

C, 100 ms mixing time.

Cordier et al.

Page 13

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 3.

HOHAHA of the Rh complex inserted into the DNA (

versus the free oligonucleotide (

The thymine H

/Me and the cytosine H

resonances are connected by solid lines. Insert:

expansion of the cytosine correlations. The loss of C2 symmetry in the central part of the

oligonucleotide is clearly apparent with the two ejected mismatched cytosines, C

a and C

which are no longer chemically equivalent. Also observable are correlations between the chrysi

protons,

(purple box). Experimental conditions: D

O, 10 °C, 100 ms mixing time.

Cordier et al.

Page 14

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 4.

2D DQF-COSY sub-spectrum of

-Rh(bpy-

)

chrysi

inserted in DNA at 10 °C (F2 × F1:

′

× H

′

-H

′′

). Only the cross-peaks associated with H1

′

-H2

′

correlations are visible. Those

corresponding to H

′

-H

′′

correlations are only observed with lower signal to noise ratio. The

cross-peak patterns indicate that all sugars, including the ejected cytosines, maintain the C

′

endo

puckering.

Cordier et al.

Page 15

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 5.

P 1D NMR of the free oligonucleotide (upper trace) and the

-Rh(bpy-

)

chrysi

inserted

in DNA (lower trace). Chemical shifts are referenced to an external standard of TMP (

= 0.00

ppm). The intense peak at

−

2.54 ppm corresponds to the phosphate buffer. All phosphate-

ester junctions in the free oligonucleotide are in the canonical B

conformation. In the Rh

complex-bound DNA, a new peak appears at

−

3.25 ppm (red arrow) corresponding to a

(

–

) of 14.7 °. This value reflects a phosphodiester linkage in the more open B

conformation.

Experimental conditions: D2O, 10 °C.

Cordier et al.

Page 16

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 6.

Structural model, adapted from the crystal structure,

illustrating the insertion of the chrysi

complex from the minor groove with ejection of the mismatched cytosines and showing metal

complex/DNA nOe’s. Observed nOe contacts are indicated with arrows.

Cordier et al.

Page 17

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 7.

NOESY sub-spectra and assignments of intermolecular contacts between the DNA and the

chrysi protons. (

) (F2 × F1: H

′

), (

) (F2 × F1: H

′

), (

) (F2 × F1: H

′

-H

′′

/Me

). The intramolecular contacts between the chrysi protons are noted

(purple line). Contacts

between the chrysi ligand and strand a (dark blue) and strand b (light green) of the

oligonucleotide are clearly separated from each other. Experimental conditions: D

O, 10 °C,

300 ms mixing time.

Cordier et al.

Page 18

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript