nihms122817.pdf

A Bulky Rhodium Complex Bound to an Adenosine-Adenosine

DNA Mismatch: General Architecture of the Metalloinsertion

Binding Mode

†

Brian M. Zeglis

Valérie C. Pierre

Jens T. Kaiser

, and

Jacqueline K. Barton

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

91125

Abstract

Two crystal structures are determined for

-Rh(bpy)

(chrysi)

(chrysi = 5,6-chrysenequinone

diimine) bound to the oligonucleotide duplex 5

′

-CGG

AATT

CCG-3

′

containing two adenosine-

adenosine mismatches (italics) through metalloinsertion. Diffraction quality crystals with two

different space groups (P3

21 and P4

2) were obtained under very similar crystallization

conditions. In both structures, the bulky rhodium complex inserts into the two mismatched sites from

the minor groove side, ejecting the mismatched bases into the major groove. The conformational

changes are localized to the mismatched site; the metal complex replaces the mismatched base pair

without an increase in base pair rise. The expansive metal complex is accommodated in the duplex

by a slight opening in the phosphodiester backbone; all sugars retain a C

′

endo

puckering, and

flanking base pairs neither stretch nor shear. The structures differ, however, in that in one of the

structures, an additional metal complex is bound by intercalation from the major groove at the central

′

-AT-3

′

step. We conclude that this additional metal complex is intercalated into this central step

because of crystal packing forces. The structures described here of

-Rh(bpy)

(chrysi)

bound to

thermodynamically destabilized AA mismatches share critical features with binding by

metalloinsertion in two other oligonucleotides containing different single base mismatches. These

results underscore the generality of the metalloinsertion as a new mode of non-covalent binding by

small molecules with a DNA duplex.

Almost fifty years ago, Lerman proposed four different non-covalent binding modes for small

molecules with DNA: (1) electrostatic binding to the sugar phosphate backbone, (2)

hydrophobic association with the minor groove, (3) intercalation into the helix by

-stacking

between adjacent base pairs, and (4) insertion into the helix by separation and displacement of

a base pair.

The first three are frequently observed and have been extensively characterized

both in solution and in the solid state.

–

In contrast, the fourth binding mode, insertion, has

eluded researchers almost completely.

Recently, however, we have structurally characterized

both by crystallography

and NMR

first examples of insertion into DNA by a small molecule,

the mismatch-specific, octahedral metal complex Rh(bpy)

(chrysi)

(chrysi = 5,6-

chrysenequinone diimine) (Figure 1).

†

Financial support for this work from the National Institutes of Health (GM33309) is gratefully acknowledged. We also thank the Gordon

and Betty Moore Foundation (Caltech Molecular Observatory). The coordinates described in this paper have been deposited in the RCSB

Protein Data Bank at the Brookhaven National Laboratory (Structure 1, RCSB ID Code: RCSB052256, PDB ID: 3GSK; Structure 2,

RCSB ID Code RCSB052255, PDB ID: 3GSJ).

*To whom correspondence should be addressed. Email: jkbarton@caltech.edu. Telephone: (626) 395-6075. Fax: (626) 577-4976.

Current address: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455.

Supporting Information. Figures showing crystal packing, DNA bending, and interduplex stacking along with Tables containing helical

parameters for structure

. This material is available free of charge via the Internet at http://pubs.acs.org.

NIH Public Access

Author Manuscript

Biochemistry

. Author manuscript; available in PMC 2010 May 26.

Published in final edited form as:

Biochemistry

. 2009 May 26; 48(20): 4247–4253. doi:10.1021/bi900194e.

NIH-PA Author Manuscript

Because insertion requires the separation of a base pair and the ejection of the bases from the

double helix, this binding mode occurs more readily at thermodynamically destabilized sites

such as single-base mismatches. Indeed, to date, insertion has only been definitively observed

with inert metal complexes bearing sterically expansive ligands, such as chrysi or phzi (benzo

[a]phenanzine-5,6-quinone diimine); in both cases, the bulky ligands are 0.5 Å wider than the

10.85 Å span of a matched AT or GC base pair.

This difference precludes the intercalation

of the complex at matched sites and thus confers specificity for binding at thermodynamically

destabilized mismatch sites.

Rhodium metalloinsertors, most notably Rh(bpy)

(chrysi)

and Rh(bpy)

(phzi)

, bind single

base mismatches with very high selectivity and with binding affinities that correlate directly

with the local destabilization associated with the single base pair mismatch.

–

This

correlation reflects the ease of separation and ejection of the mismatched bases from the double

helix. Importantly, in both cases, mismatch binding is enantiospecific: the right-handed helix

can only accommodate the right-handed (

) enantiomer. In the years since their discovery,

these metal complexes have shown significant promise not only in the detection of single base

mismatches

–

, abasic sites,

and single nucleotide polymorphisms,

but also as

chemotherapeutic agents.

–

The crystallographic structure of

-Rh(bpy)

(chrysi)

bound to a palindromic oligonucleotide

containing two CA-mismatches has recently been determined.

This structure first revealed

that the mismatch-specific rhodium complex does not bind DNA through classical

metallointercalation but rather by metalloinsertion: the complex approaches the DNA from the

minor groove side and inserts the bulky chrysi ligand at the mismatch site, extruding the

mismatched base pairs into the major groove and replacing them in the DNA

-stack. The

sugar-phosphate backbone of the DNA opens slightly to accommodate the sterically expansive

ligand at the mismatch site. Overall, the DNA is disturbed very little beyond the insertion site,

for all sugars remain in the C2

′

-endo conformation and all bases retain an

anti

configuration.

Somewhat surprising, however, was the presence of a third rhodium complex in the structure

that is bound not through insertion at the mismatch sites but through intercalation at a central

AT step. Given that no detectable binding to a matched site had been observed for these bulky

complexes in solution, we considered that this intercalation was the result of crystal packing

forces. Subsequent NMR studies of

-Rh(bpy)

(chrysi)

bound to a similar oligonucleotide

containing a CC-mismatch confirmed the insertion binding mode in solution and, significantly,

showed no evidence of an intercalated rhodium moiety.

In order to explore more generally the characteristics of the metalloinsertion mode, here we

describe two crystal structures of

-Rh(bpy)

(chrysi)

bound to an AA mismatch. Both

structures provide examples of metalloinsertion at a new mismatch, but the two structures differ

principally in the presence or absence of a third, intercalated rhodium. The comparison of these

structures with studies of the metalloinsertor bound to a CA and a CC mismatch illuminates

the general architecture of the metalloinsertion binding mode at destabilized sites in DNA.

EXPERIMENTAL

Synthesis and Purification

The metalloinsertor

–Rh(bpy)

(chrysi)

was co-crystallized with a self-complementary

oligonucleotide containing two AA mismatches (5

′

-C

-3

′

The enantiopure rhodium complexwas synthesized and isolated as described previously.

Standard oligonucleotides were synthesized from phosphoramidites on an ABI 3400 DNA

synthesizer (reagents from Glen Research) and purified both with and without the

dimethoxytrityl protecting group via two rounds of reverse-phase HPLC (HP1100 HPLC

system with Varian DynaMax

™

C18 semi-preparative column, gradient of 5:95 to 45:55

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MeCN:50 mM NH

OAc (aq) over 30 min for DMT-on purification and 2:98 to 17:83 MeCN:

50 mM NH

OAc (aq) over 30 min for DMT-off purification).

Crystal Preparation and Data Collection

Annealed oligonucleotides were incubated with the rhodium complex before crystallization.

Subsequent manipulations were performed with minimal exposure of the complex to light.

Two different sets of bright orange crystals, henceforth referred to as

and

, were obtained,

each under a distinct set of conditions. In both cases, thirteen different sequences were screened

before crystals were obtained with the sequence described above. Crystal set

was grown from

a solution of 1 mM double-stranded duplex, 3 mM enantiomerically pure

–Rh

(bpy)

(chrysi)

, 20 mM sodium cacodylate (pH 7.0), 6 mM spermine·4HCl, 40 mM NaCl,

and 5% 2-methyl-2,4-pentanediol (MPD) equilibrated in sitting drops versus a reservoir of

35% MPD at ambient temperature. The crystals grew in space group P3

21 with unit cell

dimension a = b = 48.34 Å; c = 69.50 Å,

= 90°,

= 120°, with one biomolecule per

asymmetric unit (Table 1).

Crystal set

was grown from a solution of 1 mM double-stranded duplex, 2 mM

enantiomerically pure

–Rh(bpy)

(chrysi)

, 20 mM sodium cacodylate (pH 7.0), 6 mM

spermine·4HCl, 40 mM KCl, and 5% MPD equilibrated in sitting drop versus a reservoir of

35% MPD at ambient temperature. The crystals grew in space group P4

2 with unit cell

dimensions a = b = 39.02 Å; c = 57.42 Å,

= 90°, with half of a biomolecule per

asymmetric unit (Table 1).

The data for crystal

were collected on beamline 11-1 at the Stanford Synchrotron Radiation

Laboratory (Menlo Park, CA;

= 1.00

Å, 100 K, Marrsearch 325 CCD detector). The data for

crystal

was collected from a flash-cooled crystal at 100 K on an R-axis IV image plate using

CuK

radiation produced by a Rigaku (Tokyo, Japan) RU-H3RHB rotating-anode generator

with double-focusing mirrors and an Ni filter. Both sets of data were processed with MOSFLM

and SCALA from the CCP4 suite of programs.

Crystal Structure Determination and Refinement

Both structures were solved by single anomalous dispersion using the anomalous scattering of

rhodium (f

′′

= 3.6 electrons for Rh at

= 1.54 Å, and f

′′

= 1.7 electrons for Rh at

= 1.00 Å)

with the CCP4 suite of programs. For crystal

, 2 heavy atoms were located per asymmetric

unit; for crystal

, 1.5 heavy atoms were located per asymmetric unit, with one on a special

position. Structure

was refined with PHENIX v. 1.3 against 1.6 Å data taking into account

the anomalous contribution of rhodium; for non-hydrogen atoms, anisotropic temperature

factors were refined.

The final R

cryst

and R

free

were 0.18 and 0.23, respectively. Structure

was refined using REFMAC5 v. 5.5.0066 against 1.8 Å data to a final R

cryst

= 0.18 and

free

= 0.21.

In crystal

, the rhodium complex located near the crystallographic twofold axis perpendicular

to the helical axis of the DNA intercalates in two different orientations linked by symmetry.

In crystal

, residual density with anomalous contribution was also present near a

crystallographic two-fold axis at the end of the duplex, most likely the result of disordered

cacodylate or chloride ions. In the later stages of refinement for both crystals, riding hydrogens

were included. Figures were drawn with Pymol.

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RESULTS AND DISCUSSION

Two types of crystals

The palindromic oligonucleotide 5

′

-C

-3

′

contains two

adenosine-adenosine mismatches, each situated three bases from the end of the strand and

separated from one another by a central 5

′

-AATT-3

′

step. Here, the duplex was co-crystallized

with

-Rh(bpy)

(chrysi)

for high-resolution x-ray structure determination in order to

improve our understanding of metalloinsertion at DNA single base mismatches. Interestingly,

diffraction quality crystals with two different space groups (P3

21 and P4

2) were obtained

under very similar crystallization conditions. Indeed, both crystals were grown with the same

temperature, buffer, pH, type and concentration of precipitant, concentration of DNA, and

concentration of spermine. The only differences are the concentration of metalloinsertor and

the identity of salt employed: crystal

(P3

21), containing 2 rhodiums per duplex, was obtained

using 3 mM complex and 40 mM NaCl, and crystal

(P4

2), containing 3 rhodiums per

duplex, was obtained using 2 mM metalloinsertor and 40 mM KCl. Taken together, the

structures of crystal

(1.6 Å) and

(1.8 Å) provide insights into the structure and generality

of metalloinsertion.

Structure 1

In crystal

, the oligonucleotide co-crystallizes with the metalloinsertor in the space group

21, with six asymmetric units per unit cell. The asymmetric unit contains one DNA duplex

complexed with two metalloinsertors (Figure 2). Significantly, crystallization breaks the C

symmetry of the DNA-metalloinsertor palindromic assembly, rendering the two mismatch sites

inequivalent and providing two independent views of the mismatched site. Inspection of the

unit cell reveals that the duplexes do not stack head-to-tail to form a longer double helix, as is

frequently observed with DNA. Instead, it is the inter-duplex

-stacking of the ejected

adenosines, either interwoven with the ancillary bpy ligand of a nearby rhodium moiety or

stacked with adjacent, ejected adenosines, that determines the overall crystal packing and thus

the space group.

The two mismatched sites, not related by symmetry, provide separate views of the

metalloinsertion. In both cases, the metal complex inserts from the minor groove by separating

and ejecting the mismatched bases. The sterically expansive chrysi ligand of the metalloinsertor

replaces the destabilized bases in the helical

-stack. The two ejected purines are pushed

outward into the major groove. One of them remains close and perpendicular to the base stack,

while the other folds back to the minor groove in a position stabilized by crystal packing. In

both cases, deep insertion in the double helix is not inhibited by the increased steric hindrance

of the minor groove: the distance between the rhodium center and the helical axis is 4.8 Å,

approximately half the radius of the duplex.

Upon binding, the rhodium complex inserts deeply to enable complete overlap and stacking

with both the purines and pyrimidines of the flanking base pairs. Importantly, these flanking

base pairs neither stretch nor shear despite the considerable width of the ligand. All sugars

retain their original C

′

endo

puckering, and all bases maintain their initial

anti

conformation.

To accommodate the inserted rhodium complex, the minor groove at the binding site widens

to 19 Å from phosphate to phosphate, between 1 and 1.5 Å wider than other points in the duplex.

Aside from the opening of the phosphodiester junctions at the insertion site, however, very

little distortion of the DNA is observed (Tables 2 and 3).

The difference between the two insertion sites lies only in the crystal packing of the ejected

adenosines. In one of the two insertion sites, one of the ejected adenosines is stacked tightly

within the major groove, where it lies perpendicular to the DNA base stack and is not involved

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in any interduplex interactions or hydrogen-bonding (Supporting Information). In contrast, the

other adenosine at this site is interwoven with and

-stacks between the ejected adenosine from

an adjacent duplex and the ancillary bipyridine ligand of the rhodium complex inserted in that

nearby oligonucleotide. At the second insertion site, one of the ejected adenosines again

stacks between the ejected adenosine from a second adjacent oligonucleotide and the ancillary

bipyridine of the rhodium complex intercalated in that nearbyDNA. Unlike at the first insertion

site, however, the other ejected adenosine here does partake in

-stacking, in this case with an

extruded adenosine of yet another nearby duplex (Figure 3).

Structure 2

In crystal

, the oligonucleotide co-crystallizes with the metalloinsertor in the space group

2. In this case, the asymmetric unit is a single DNA strand with 1.5 metalloinsertors.

Each duplex thus contains three rhodium complexes, one inserted at each of the mismatched

sites and a third intercalated between the adenosine and thymine of the central 5

′

-AT-3

′

step

(Figure 4). Due to its position on a crystallographic two-fold axis, the central rhodium

intercalates in two different orientations. The rhodium complexes at the two mismatched sites

are also related by C

symmetry, providing a single, independent view of the insertion site.

Interestingly, in all respects other than the identity of the mismatch, this structure is virtually

identical to that previously published for

-Rh(bpy)

(chrysi)

bound to a CA mismatch.

At the AA mismatch site, the metalloinsertor approaches the DNA from the minor groove,

ejects the mispaired adenines from the helix, and replaces them in the DNA base stack with its

own sterically expansive chrysi ligand. Indeed, the metalloinsertor

-stacks with the flanking

AT and CG base pairs and penetrates so deeply from the minor groove that it is solvent

accessible from the major groove. One of the ejected adenosines sits in the major groove,

positioned perpendicular to the DNA base stack. The other adenosine bends back into the minor

groove, where it

-stacks between the ejected adenosine of an adjacent duplex and a bipyridine

ligand of a metalloinsertor bound to that oligonucleotide. Insertion of the rhodium complex

into the site is facilitated by a slight widening of the phosphate backbone, from an average of

17.5 Å for well-matched sites to 19 Å for the metalloinsertion sites. Indeed, beyond this

conformational change, metalloinsertion again distorts the DNA very little. Some buckling of

the external flanking CG basepair is observed, but all riboses exhibit C2

′

endo

puckering, and

all bases retain an

anti

configuration (Supporting Information).

As in the CA-mismatch structure,

a third

-Rh(bpy)

(chrysi)

is also found intercalated at

the central 5

′

-AT-3

′

step. At this site, the rhodium complex approaches the duplex from the

major groove and intercalates the chrysi ligand between adjacent AT and TA base pairs,

doubling the rise at the intercalation site to 7.1 Å and slightly unwinding the duplex (Supporting

Information). This binding interaction resembles closely that previously observed in the crystal

structure of the sequence-specific metallointercalator

-Rh[(R,R)-Me

trien](phi)

bound

by classical intercalation to its target site.

The intercalative binding, like insertion, is

accommodated by a slight widening of the phosphate backbone at the intercalation site and is

accompanied by some buckling of the adjacent base pairs. Given the exquisite mismatch

selectivity of the metalloinsertors in solution, such intercalative binding is a surprise and is

almost certainly the result of crystal packing forces. The bipyridines of the intercalated metal

complex

-stack with the terminal CG base pairs of two crystallographically related duplexes,

in essence making the intercalated rhodium complex a linchpin for the crystal packing

(Supporting Information).

Differences between the two structures

Certainly the telling difference between the two structures is the presence or absence of a

Rh(bpy)

(chrysi)

intercalated at the central 5

′

-AT-3

′

step. Given the similarity in

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crystallization conditions for crystals

and

, the rhodium complex likely has comparable

affinity for this central matched site in both cases. That the intercalated rhodium complex

not

observed in structure

therefore strongly substantiates our conclusion that

-Rh

(bpy)

(chrysi)

has negligible affinity for matched DNA and only binds to such sites when

intercalation is stabilized by crystal packing-driven

-stacking. In structure

and the previously

reported CA-mismatch structure,

intercalation at the matched site is supported by

-stacking

between the ancillary bipyridines of the intercalated rhodium complex and the terminal CG

base pairs of two adjacent helices. Moreover, interwoven stacking between rhodium moieties

in these latter duplexes and ejected purines further serves to lock the helices in an orientation

that favors intercalative binding. These interactions, taken together, promote the binding of the

metalloinsertor in a mode that is not detectable in solution. In fact, the interactions are

insufficient to enforce complete intercalation into the double helix (the Rh-helical axis distance

in the CA-structure, for example, is 1.24 Å longer than in the case of the DNA-bound

metallointercalator

-Rh[(R,R)-Me

trien](phi)

). These structures, taken together, provide

a cautionary example of how crystal packing forces may alter the binding of small molecules

with DNA.

The intercalated

–Rh(bpy)

(chrysi)

in structure

is likely also responsible for a second

major difference between the structures. Upon superposition of the two structures, it becomes

evident that the duplex in structure

is slightly bent relative to that in structure

(Supporting

Information). Examination of the two mismatch-bound chrysi ligands in each structure is

particularly instructive in this regard: in structure

, the two ligands are nearly coplanar,

whereas in structure

, they are clearly skewed relative to one another. Because few

perturbations to the duplex are observed beyond the mismatched base pair itself in either

structure, it is improbable that the metalloinsertors are responsible for this bend in the duplex.

Rather, the slight bending is most likely a result of the flexibility associated with the base step.

It follows that in structure

, the centrally intercalated and well-stacked rhodium complex

rigidifies and straightens the helix.

A third major difference between the two structures lies in the stacking of the extrahelical

adenosines. The interduplex, four component

-stacking interactions of one of the ejected

adenosines at each mismatch site is common to both structures reported here, as well as the

previously published CA-mismatch structure. It is with the second ejected base at each

mismatch site that differences arise. At each AA-mismatch site in structure

and in the CA-

mismatch structure, the second ejected adenine or ejected cytosine, respectively, sits tightly

within the major groove, perpendicular to the DNA base stack and uninvolved in any

-stacking

or hydrogen bonding. The same is true for the second ejected adenine at one of the two AA-

mismatch sites in structure

. At the other AA-site in structure

, however, the second ejected

adenine lies near the major groove, remains close to the phosphate backbone, and

-stacks with

the ejected adenine of a nearby duplex (Figure 3).

General architecture of the insertion binding mode

What is perhaps most remarkable about these crystal structures is not their differences but their

similarity, not only to one another but also to the earlier structure we obtained.

The

superposition of the four independent views of

-Rh(bpy)

(chrysi)

bound to a mismatch site

(3 AA-mismatch sites, 1 CA-mismatch site) reveals how every detail of the insertion binding

mode is maintained regardless of the type of mismatch (Figure 5). In all cases, the DNA

conformational changes are localized to the binding site. The metal complex essentially

replaces the mismatched base pair; there is no increase in rise, no change in stacking, and no

change in sugar puckering. In every case,

Rh(bpy)

(chrysi)

is well stacked with the

matched DNA bases and penetrates the DNA so deeply that it protrudes from the opposite

major groove. Furthermore, in each study, this binding is accommodated by a slight opening

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in the phosphodiester backbone, and the DNA is only minimally perturbed beyond the insertion

site: all bases maintain their original

anti

conformation, all sugars retain a C

′

endo

puckering,

and flanking base pairs neither stretch nor shear. Perhaps most remarkable is that even the

positions of the ejected bases, irrespective of their identities, assume nearly identical positions.

The ejected bases are not splayed out in random positions, at least not in the structures in the

solid state. Instead, the positions of the ejected bases seem to be defined, at least in part, by the

sugar torsions. In fact, it may be more facile for the bases to be ejected from the minor groove

side and accommodated in the major groove; this ejection into the major groove may then be

a general characteristic of base pair displacement.

Certainly, as evident in Figure 5, the

distinct overlap of these different insertion sites, independent of the mismatch identity and

crystal packing, must reflect the ease of adopting this conformation. These results, all taken

together, indicate clearly that insertion into the double helix from the minor groove side with

ejection of a base pair towards the major groove is a motif that is characteristic of binding of

metal complexes bearing extended ligands to thermodynamically destabilized sites in DNA.

CONCLUSIONS

The metalloinsertion of bulky metal complexes at DNA mismatches represents a new paradigm

for how small molecules may bind non-covalently to the DNA duplex. The structures described

here of

-Rh(bpy)

(chrysi)

bound to thermodynamically destabilized AA-mismatches

illustrate the generality of this binding mode. Combined with previous crystallographic

and

NMR

studies on different mismatched oligonucleotides, these structures reveal the

architectural characteristics of metalloinsertion: in every case, without regard to the type of

mismatch, the metal complex approaches the DNA from the minor groove, ejects the

mismatched bases from the helix towards the major groove, replaces the extruded pair in the

base stack with its own bulky ligand, and perturbs the DNA only minimally beyond the local

binding site. The similarity in structures described here along with their clear differences serves

furthermore to underscore metalloinsertion as a unique binding interaction, one distinct from

intercalation. The presence of an intercalative rhodium in one of the structures also highlights

how crystal packing forces can contribute to the solid state structures of small molecules bound

non-covalently to DNA. While the information obtained from these structures yields critical

and detailed insights, these data must also be considered in context with other data obtained in

solution. In future work, it is hoped that these structures will not only prove useful as an

illustration of a binding archetype but also in driving the design, synthesis, and application of

new generations of small molecules that bind DNA through the insertion mode.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank Dr. Leonard Thomas for valuable discussions.

Abbreviations

chrysi

chrysene-5,6-quinone diimine

phzi

benzo[a]phenazine-5,6-quinone diimine

DMT

dimethoxyltrityl

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MPD

2-methyl-2,4-pentanediol

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16637630]

17. Zeglis BM, Boland JA, Barton JK. Targeting Abasic Sites and Single Base Bulges in DNA with

Metalloinsertors. J Am Chem Soc 2008;130:7530–7531. [PubMed: 18491905]

18. Zeglis BM, Boland JA, Barton JK. Recognition of Abasic Sites and Single Base Bulges in DNA with

a Metalloinsertor. Biochemistry ASAP. 2009

19. Hart JR, Johnson MD, Barton JK. Single-nucleotide polymorphism discovery by targeted DNA

photocleavage. Proc Nat Acad Sci USA 2004;101:14040–14044. [PubMed: 15383659]

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DNA Alkylating Agents. J Am Chem Soc 2004;126:8630–8631. [PubMed: 15250697]

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and Applications. Nat Prot 2007;2:357–371.

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Zeglis et al.

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Biochemistry

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Figure 1.

-Rh(bpy)

(chrysi)

Zeglis et al.

Page 10

Biochemistry

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NIH-PA Author Manuscript

Figure 2.

Structure

: two

-Rh(bpy)

(chrysi)

(red) are inserted, one in each AA mismatch of the

oligonucleotide 5

′

-CGGAAATTACCG-3

′

(green). The ejected adenosines are shown in blue.

Zeglis et al.

Page 11

Biochemistry

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NIH-PA Author Manuscript

Figure 3.

Crystal packing by the ejected adenosines at one of the metalloinsertion sites in structure

. At

both insertion sites of the duplex, one ejected adenosine (cyan)

-stacks in an interwoven

fashion with the bipyridine ligand of a rhodium complex (yellow) inserted in a nearby

crystallographically related oligonucleotide and its corresponding ejected adenosine (red). The

bipyridine ligand of the rhodium complex in the original duplex (green) completes the four-

component stacking. In only one of the two insertion sites, as shown here, the second

mismatched adenosine ejected in the major groove (magenta)

-stacks with a

crystallographically equivalent ejected major groove adenosine (blue).

Zeglis et al.

Page 12

Biochemistry

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NIH-PA Author Manuscript

Figure 4.

Structure

two

-Rh(bpy)

(chrysi)

(red) are inserted, one in each AA mismatch of the

oligonucleotide 5

′

-CGGAAATTACCG-3

′

(purple). A third rhodium complex (blue) is

intercalated at the central 5

′

-AT-3

′

step. The ejected adenosines are shown in green.

Zeglis et al.

Page 13

Biochemistry

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NIH-PA Author Manuscript

Figure 5.

Superposition of the three crystal structures showing insertion of

-Rh(bpy)

(chrysi)

into a

single base mismatch viewed looking into the major groove (left) or minor groove (right). The

red, blue, and orange structures represent insertion into an AA mismatch as reported in this

work (red and blue are the two sites from structure

, and orange is from structure

). The cyan

structure represents insertion into a CA mismatch as previously reported.

Zeglis et al.

Page 14

Biochemistry

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Zeglis et al.

Page 15

Table 1

Data collection and refinement statistics

Structure 1

Structure 2

Data Collection

Space group

Cell dimensions:

a, b, c

48.3, 48.3, 69.5

39.0, 39.0, 57.4

90.0, 90.0, 120.0

90.0, 90.0, 90.0

Wavelength

1.0046

1.5418

Resolution

35.0-1.60 (1.69-1.60)

28.71- 1.80 (1.90-1.80)

merge

0.035 (0.499)

0.061 (0.782)

pim

0.013 (0.288)

0.031 (0.342)

26.7 (2.0)

19.1 (2.3)

Completeness, %

99.5 (98.9)

98.7 ( 97.4)

Redundancy

7.9 (4.2)

6.5 ( 6.6)

Refinement

No. of Reflections

22677

4469

work

free

0.184/0.227

0.183/0.213

No. of atoms (DNA)

524

262

No. of atoms (RhL

)

120

No. of atoms (water)

B-factors (DNA)

43.44

25.7

B-factors (complex)

43.44

22.1

B-factors (water)

48.86

41.4

RMS dev. (lengths)

0.013

0.032

RMS dev. (angles)

2.450

4.281

Biochemistry

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Zeglis et al.

Page 16

Table 2

DNA helical parameters

relating consecutive base pairs of structure

Base-pair

Shift (Å)

Slide (Å)

Rise (Å)

Tilt (°)

Roll (°)

Twist (°)

CG/CG

0.8

2.2

3.4

12.0

−

1.6

37.6

GG/CC

−

0.3

2.7

3.2

−

6.1

5.7

34.3

GA/TC

AA/TT

−

1.3

1.2

3.3

−

4.7

3.8

37.6

AT/AT

0.0

0.1

3.4

1.3

−

0.7

29.6

TT/AA

1.3

1.0

3.4

2.2

5.6

36.1

TC/GA

CC/GG

0.4

2.7

3.3

4.7

6.5

34.5

CG/CG

−

1.0

2.5

3.2

−

8.2

2.1

37.3

B-DNA

−

0.1

−

0.8

3.3

−

1.3

−

3.6

Geometrical relationships between consecutive base pairs: shift, translation into the groove; slide, translation toward the phosphodiester backbone; rise, translation along the helix axis; tilt, rotation

about the pseudo-two-fold axis relating the DNA strands; roll, rotation about a vector between the C1

′

atoms; and twist, rotation about the helix axis.

Data were calculated by using the program 3DNA.

Biochemistry

. Author manuscript; available in PMC 2010 May 26.

NIH-PA Author Manuscript

Zeglis et al.

Page 17

Table 3

DNA helical parameters for the base pairs of structure

Base-pair

Shear (Å)

Stretch (Å)

Stagger (Å)

Buckle (°)

Propeller (°)

Opening (°)

Sugar pucker

C-G

0.1

−

0.1

0.9

−

10.0

−

2.5

−

2.8

′

-endo

G-C

−

0.2

−

0.1

−

0.0

−

0.2

1.1

−

3.8

′

-endo

G-C

−

0.4

−

0.1

0.6

15.0

−

7.2

−

1.3

′

-endo

A-A

′

-endo

A-T

−

0.1

−

0.1

0.0

−

8.6

7.2

1.5

′

-endo

A-T

0.1

−

0.1

−

0.0

−

8.3

3.2

′

-endo

T-A

−

0.0

−

0.0

−

0.8

−

8.0

1.1

′

-endo

T-A

−

0.1

−

0.2

0.1

5.8

9.4

1.7

′

-endo

A-A

′

-endo

C-G

0.3

−

0.1

0.6

−

18.8

−

5.8

−

0.5

′

-endo

C-G

0.2

−

0.1

−

3.9

2.0

−

0.5

′

-endo

G-C

−

0.3

−

0.1

0.4

6.2

−

5.3

0.7

′

-endo

B-DNA

0.1

4.1

−

4.1

′

-endo

Data were calculated by using the program 3DNA.

Biochemistry

. Author manuscript; available in PMC 2010 May 26.