nihms122913.pdf

DNA base mismatch detection with bulky rhodium intercalators:

synthesis and applications

Brian M. Zeglis

and

Jacqueline K. Barton

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

91125

Abstract

The syntheses and applications of two metallointercalators, Rh(bpy)

(chrysi)

and Rh

(bpy)

(phzi)

, that target single base mismatches in DNA are described. The complexes selectively

bind thermodynamically destabilized mismatched DNA sites and, upon photoactivation, promote

strand scission neighboring the mismatch. Owing to their high specificity, independent of sequence

context, targeting mismatches with these complexes offer an effective and convenient alternative to

current mismatch- and SNP-detection methodologies. Syntheses of these complexes are described

as well as protocols for the application of these complexes in marking mismatched sites. In the first,

the irradiation of [

P]-labeled duplex DNA with either intercalator followed by denaturing PAGE

allows for the detection of mismatches in oligonucleotide sequences. The second protocol describes

a method for the efficient detection of single nucleotide polymorphisms (SNPs) in larger genes or

plasmids. Pooled genes are denatured and re- annealed to form heteroduplexes, incubated with Rh

(bpy)

(chrysi)

or Rh(bpy)

(phzi)

, irradiated, and analyzed by capillary electrophoresis for

fragments indicative of mismatch and thus SNP sites. The synthesis of the metallointercalators

requires about five to seven days. Both the mismatch and SNP detection experiments require

approximately three days.

INTRODUCTION

The synthesis and study of octahedral metal complexes that bind DNA have long been pursuits

of our laboratory

. The unique modularity of metal complexes has allowed us to investigate

systematically the factors that contribute to DNA site recognition. For example, the site

selectivity of rhodium complexes bearing the 5,6-phenanthrenequinone diimine (phi)

intercalating ligand varies dramatically with the identity of the ancillary ligands: Rh

(bpy)

(phi)

shows little site selectivity while Rh(R,R-dimethyltrien)(phi)

binds

specifically to 5

′

-TGCA-3

′

sites

In recent years, we have applied our understanding of molecular recognition elements to the

development of complexes that selectively bind mispaired sites in DNA

. DNA mismatches

occur in the cell as a result of polymerase errors or DNA damage

. In order to preserve the

fidelity of its genome, the cell has developed a complex mismatch repair (MMR) machinery

to find and correct these mismatches, thus preventing consequent mutations

. Abnormalities

in this machinery, however, can lead to the accumulation of mismatches, and thus mutations,

in the genome with a likelihood of cancerous transformation. Indeed, mutations in MMR genes

have been identified in 80% of hereditary non-polyposis colon cancers, and 15–20% of biopsied

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

COMPETING FINANCIAL INTERESTS:

The authors have no competing financial interests regarding this chemistry.

NIH Public Access

Author Manuscript

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

Published in final edited form as:

Nat Protoc

. 2007 ; 2(2): 357–371. doi:10.1038/nprot.2007.22.

NIH-PA Author Manuscript

solid tumors have shown evidence of somatic mutations associated with MMR

Mismatch detection may provide an early indicator of these cancerous transformations

Given the wealth of sequence information available through the human genome project, much

attention has turned to the discovery of single nucleotide polymorphisms (SNPs), the single

base differences that lead to variations in one’s disposition to disease or response to

pharmaceuticals

. Importantly, DNA mismatches can also serve as important intermediates

in the search for SNPs. When a test gene fragment is mixed with the wild type fragment, and

the DNA heated and reannealed, 50% of the reannealed duplexes will contain single base

mismatches if the test DNA contains a SNP. These mismatches thus provide the target for SNP

detection

. Figure 1 schematically illustrates how mismatch targeting can be usefully applied

in both situations: the discovery of SNPs and the detection of deficiencies in MMR.

In our laboratory, two families of mismatch-specific metallointercalators have been developed

based on a pair of bulky intercalating ligands, 5,6-chrysenequinone diimine (chrysi) and 3,2-

benzo[a]phenazine quinone diimne (phzi) (Fig. 2)

. The preparation of the simplest

complexes in each family, Rh(bpy)

(chrysi)

and Rh(bpy)

(phzi)

, are described here. In

both cases, the sterically expansive ligand is too large to intercalate easily into the base stack

of regular B-form DNA. However, each binds with high affinity to the thermodynamically

destabilized mismatched sites. Binding affinities are on the order of 10

−

for Rh

(bpy)

(chrysi)

and 10

−

for Rh(bpy)

(phzi)

10,13. Importantly, affinities correlate with

the destabilization associated with a mismatch. Our structural model is shown in Figure 3. The

correlation between affinity and mismatch stabilization can be understood based upon the ease

of extruding the mismatched bases upon insertion of the metal complex into the base pair stack

(Pierre, Kaiser, Barton submitted). Those sites that are most destabilized are most easily bound

by the metal complexes. In all, the compounds bind > 80% of mismatch sites in all possible

sequence contexts

Despite their differences in binding strength, both complexes exhibit

≥

1000-fold selectivity

for mismatched DNA sites over Watson-Crick base paired DNA sites. Site-selectivity in

solution can be readily discerned through DNA photocleavage experiments. In addition to

tightly and selectively binding mismatches, the complexes promote direct strand scission

adjacent to the mismatch site with photoactivation

. The selectivity of the complexes is a

tremendous asset; Rh(bpy)

(chrysi)

, for example, is capable of binding and cleaving a single

CC mismatch in a 2725 base pair plasmid

. Because the complexes target only those sites that

are thermodynamically destabilized in the base stack, however, more stable mismatches, for

example, those containing guanines, are not readily detected based upon photocleavage.

Interestingly, the

-enantiomers of the complexes bind far better than the

-enantiomers. Thus,

while racemic complexes may be used for all the experiments described here, only the

enantiomer is required.

This protocol first describes the synthesis of Rh(bpy)

(chrysi)

and Rh(bpy)

(phzi)

and the

enantiomeric separation of

- and

-Rh(bpy)

(chrysi)

16. First explained are the syntheses

of the two intercalating ligands (Fig. 4 and 5). The two compounds, then, are synthesized

identically from RhCl

until the last step, in which the ligand diones are condensed onto Rh

(bpy)

(NH

)

under basic conditions (Fig. 6). It should be pointed out that other methods

for the formation of the diimine complexes are known. All others, however, require the

cumbersome anaerobic coordination of the ligand diamine followed by the subsequent

oxidation of the diamine to the diimine;

this alternative methodology is further limited by

low product yields and the necessity of using the ligand diamine precursor. In contrast, the

condensation method we employ is far more synthetically facile and produces the desired

products in high yield. The UV-visible spectra for the complexes are given in Figure 7. The

enantiomeric separation of

- and

-Rh(bpy)

(chrysi)

is achieved through the somewhat

Zeglis and Barton

Page 2

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

unorthodox use of 0.15 M (+)-potassium antimonyl tartrate as a chiral eluant for a cation

exchange column (Fig. 8). Other methods, including HPLC using a chiral column, have been

attempted but have yielded poorer enantiomeric separation. The circular dichrosim spectra are

provided in Figure 9.

In addition to the syntheses and enantiomeric separation, two experimental applications of the

mismatch-selective complexes are included. In the first, a PAGE sequencing gel can be used

to visualize the presence and location of mismatched photocleavage sites in oligonucleotide

DNA sequences. In this method, samples of duplex DNA, in which a very small amount of

one of the strands has been 5

′

P]-labeled, are irradiated in the presence of Rh

(bpy)

(chrysi)

or Rh(bpy)

(phzi)

. The radioactive samples are then eluted through a

denaturing polyacrylamide gel and visualized via phosphorimagery. If the original duplex

strand contains mismatches, bands corresponding to shorter fragments created by

photocleavage at the mismatched site are evident in addition to the full-length parent band (see

Fig. 10 for a sample PAGE experiment). While this methodology may be somewhat limited

by the DNA length, it provides information on both the presence (or absence) of a mismatched

site and, with the help of parallel sequencing ladders, the exact location of the mismatched site

in the sequence

. Of course, other procedures have been developed to search DNA for

mismatches using enzymatic or chemical methods, such as RNAase cleavage and chemical

cleavage with osmium tetroxide or hydroxylamine

. However, none of these combines the

accuracy, selectivity, robustness, and ease desired in a clinical testing procedure.

The mismatch-specificity of our complexes can also be used to detect single nucleotide

polymorphisms (SNPs)

. In this second application, a regions of the genome, perhaps

containing an SNP, is amplified, denatured, and re-annealed in the presence of a pooled sample

to create heteroduplexes that may contain a mismatch at the site of the original SNP. After

annealing, the samples are incubated with Rh(bpy)

(chrysi)

or Rh(bpy)

(phzi)

, irradiated

to promote photocleavage, end-labeled with a fluorophore, and analyzed by capillary gel

electrophoresis. If an SNP is present in one of the original segments amplified, the capillary

electrophoresis trace will display both a parent peak for the full, uncleaved strand and a peak

that corresponds to a fragment resulting from photocleavage at the mismatched site (see Fig.

11 schematic of the procedure). Importantly, this technique allows for the detection of SNPs

with allele frequencies as low as 5% from a single pooled sample. This method greatly reduces

the cost of discovering new SNPs. Perhaps the greatest advantage of our method is the ease of

use; specifically, fewer polymerase chain reactions (PCRs), and no cycle sequencing are

required.

An alternative method for SNP detection, resequencing, is known

–

. However,

resequencing is expensive with respect to materials, labor, and data processing. Further, while

the current technique must scan a particular region of the genome many times to detect a single

SNP, even then the false positive rate is high

. Resequencing does, however, hold certain

advantages over our method, namely that it can provide a clear picture of both the sequence

identity of the SNP and its frequency. We recommend using first our SNP protocol for

discovery followed by resequencing of the specific region marked by the metal complex for

characterization. Perhaps, then, in an ideal situation metallointercalators and resequencing

could be used in concert to discover new SNPs and create extensive genomic SNP maps.

The possible uses of our mismatch-specific metallointercalators extend well beyond the two

applications outlined here. For example, trisheteroleptic complexes bearing both a mismatch-

binding intercalating ligand and a linker-modified bipyridine ligand have been synthesized for

the development of mismatch-directed bifunctional conjugates. To date, this strategy has been

employed to make bifuvntional conjugates for platination

and alkylation

near mismatched

sites as well as conjugates for fluorescence-based mismatch detection

, and to improve nuclear

Zeglis and Barton

Page 3

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

uptake of the mismatch-specific complexes

. Moreover, recent experiments have revealed

that Rh(bpy)

(chrysi)

and Rh(bpy)

(phzi)

may have chemotherapeutic value. Both

complexes have been shown to inhibit selectively the cell proliferation of MMR-deficient cells

over MMR-proficient cells

MATERIALS

REAGENTS

•

Chrysene (Aldrich Zone Purified)

•

Glacial acetic acid

•

Sodium dichromate (Aldrich)

•

2,3-Dichloro-1,4-napthoquinone (Aldrich)

•

o-Phenylene diamine (Fluka)

•

Pyridine

•

Concentrated nitric acid (not fuming yellow or red)

•

Water (deionized and filtered, i.e. Millipore purified to > 18

resistivity)

•

Ethanol

•

Diethyl ether

•

RhCl

hydrate (Pressure Chemicals)

•

Hydrazine monohydrate (Aldrich)

•

2,2

′

-Bipyridyl (Aldrich)

•

Triflic acid (5g ampule, Fluka)

•

Concentrated ammonium hydroxide

•

Sodium hydroxide

•

Acetonitrile

•

Methanol

•

Trifluoroacetic acid (TFA)

•

Potassium nitrate (Aldrich)

•

Sephadex

SP-C25 ion exchange resin (Aldrich)

•

Magnesium chloride

•

(+)-KSb-tartrate (Aldrich)

•

Sodium chloride

•

Sodium phosphate (NaH

, referred to as NaPi)

•

Polynucleotide kinase (Roche, 10 activity units/

•

Polynucleotide kinase buffer (Roche, supplied with PNK)

•

[

P]-ATP (End-labeling grade, MP Biomedicals)

•

10X Tris-borate-EDTA (TBE) buffer (National Diagnostics)

•

Ultrapure SequaGel

Sequencing System Diluent (National Diagnostics)

Zeglis and Barton

Page 4

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

•

Ultrapure SequaGel

Sequencing System Concentrate (National Diagnostics)

•

Ultrapure SequaGel

Sequencing System Buffer (National Diagnostics)

•

Ultrapure TEMED (Invitrogen)

•

Ammonium persulfate (MP Biomedicals)

•

Denaturing formamide loading dye (see

REAGENT SET-UP)

•

Saran Wrap

(or equivalent product)

•

Exo

I (New England Biolabs)

•

Calf Intestinal Alkaline Phosphtase (Roche)

•

QIAquick

PCR purification kit (Qiagen)

•

Tris Hydrochloride

•

Dithioerythritol

•

Xho

I (Roche)

•

Cla

I (Roche)

•

SNaPshot

Fluorescent Labeling Kit (Applied Biosystems)

•

Chloroform

•

Ethyl acetate

•

Magnesium chloride

EQUIPMENT

•

Assortment of round-bottom flasks (50 mL, 100 mL, 250 mL, 500 mL)

•

Reflux condensers (14/20 and 24/40 joints)

•

Magnetic stir bar and stir plate

•

Heating mantle

•

500 mL Beaker

•

60 mL Medium glass frit

•

500 mL Vacuum flask

•

50 mL Schlenk flask

•

Pasteur pipettes

•

Silica TLC plates (Baker)

•

Rotary evaporator

•

Flash column (about 20 cm × 1.5 cm)

•

Very long glass column (about 1.7 m × 1.5 cm)

•

2 Medium-sized glass columns (about 0.5 m × 1.5 cm)

•

Tubing (Tygon)

•

Lyophilizer

•

Razor blades

Zeglis and Barton

Page 5

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

•

Tweezers

•

X-ray film (Kodak BioMax XAR film, 35 cm × 43 cm)

•

X-ray developer (i.e. Konica Minolta SRX-101A)

•

1.7 mL Centrifuge tubes

•

Microcentrifuge (i.e. Eppendorf 5415C)

•

Lucite sample boxes for 1.7 mL centrifuge tubes

•

Reverse phase C18 cartridges (i.e. Waters Sep-Pak

)

•

Micro Bio-Spin

6 Chromatography Column (Bio-rad)

•

Evacuated centrifuge (SpeedVac)

•

Glass gel plates, notched and un-notched (35 cm × 45 cm, FisherBiotech)

•

Gel running apparatus (23 cm × 40 cm × 50 cm, FisherBiotech)

•

Power supply for gel running apparatus (Fisher Biotech, FB-EC-4000P)

•

Adjustable heat block

•

Scintillation counter

•

Assortment of micropipettes (10

L, 20

L, 200

L, 1000

•

Light source (see

EQUIPMENT SET-UP

)

•

Phosphorimager screen (35 × 43 cm, Molecular Dynamics)

•

Phosphorimager (Storm 820, GE Healthcare)

•

Photoshop Software (Adobe)

•

ImageQuant Software (Molecular Dynamics)

•

Thermocycler

•

Peristaltic Pump

REAGENT SET-UP—

Denaturing Formamide Loading Dye: 80% formamide, 10 mM

NaOH, 0.025% xylene cyanol, 0.025% bromophenol blue, in 1X TBE buffer.

EQIPMENT SET-UP—

Both Rh(bpy)

(chrysi)

and Rh(bpy)

(phzi)

cleave DNA at a

variety of wavelengths; while this allows for a number of light source options, the cleavage

efficiency is highly wavelength-dependent. Typically, irradiations are performed using either

a solar simulator (Oriel Instruments, wavelength output 320–450 nm) equipped with a UV

filter or at 340 or 442 nm on a 1,000 W Hg/Xe arc lamp equipped with a monochromator, a

295-nm UV-cutoff filter, and an IR filter (Oriel Instruments). Typical irradiation times are 15

min on the solar simulator and 30–60 min on the lamp. Other options exist, however. Cleavage

can also be induced using a 302-nm transilluminator or a 365-nm “black-light” situated 3–4

cm above an open sample tube. However, in these two cases, about 10X more irradiation time

(when compared to the more powerful lamp and solar simulator) is required to prompt

substantial photocleavage. Neither white fluorescent light nor incandescent light provide

sufficient UV power for strand cleavage with Rh(bpy)

(chrysi)

or Rh(bpy)

(phzi)

Regardless of the source, all irradiations are performed in 1.7 mL centrifuge tubes. In a case

where the light shines vertically from above, transparent Lucite

samples boxes are simple

and effective sample holders. In a case where the light shines horizontally, sample holders

more typical of laser set-ups can be employed.

Zeglis and Barton

Page 6

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

While other options exist for capillary electrophoresis, this procedure has been optimized for

the use of an ABI Prism 310 instrument (Applied Biosystems). A sample instrument

configuration is shown below:

capillary: 47 cm × 50

SNaPshot dye: Hex

polymer: POP-4

dye set: E5

molecular weight marker: Genescan 500 TAMRA

module: GS STR POP4 (1 mL)

injection seconds: 5 seconds

injection kV: 15.0 V

run kV: 15.0 V

run temperature: 60°C

run time: 35 min

analysis matrix: matrix E5

analysis size standard: gs-500liz

analysis parameters: 0–500 base pairs

PROCEDURE

General note of caution for syntheses

5,6-chrysene quinone, 3,2-benzo[a]phenazine quinone, and their metal complexes are DNA

intercalators. While their exact toxicity profile is unknown, they are almost certainly highly

toxic and most likely carcinogenic. These compounds should be handled with extreme care.

Synthesis of 5,6-chrysene quinone

1 Combine 10.0 grams (44 mmol) of chrysene and 110 mL of glacial acetic acid in a 250

mL round bottom flask and stir vigorously.

CAUTION:

Chrysene is a known carcinogen.

2 Slowly add 46 g sodium dichromate to the stirring slurry.

3 Affix a reflux condenser and heat the slurry to reflux for 24 h. Over the course of the

heating the color of the solution will slowly change from bright orange with white solid

to dark green/brown with orange precipitate.

CRITICAL STEP:

The reaction can be monitored by color change. The reaction is

complete when no more white solid can be seen in the refluxing mixture.

4 After 24 h, stop heating. Remove the reflux condenser once boiling has stopped, and

before the mixture reaches room temperature, pour it into 100 mL of rapidly boiling water

in a beaker.

5 While still hot, filter the solution through a medium glass frit.

CAUTION:

Hot-filtering, while necessary in this case, is always dangerous. Be careful.

6 Wash the collected red solid 3 times with 100 mL boiling water.

Zeglis and Barton

Page 7

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

CRITICAL STEP:

The filtration MUST be performed when the solution is boiling or

near-boiling. Any cooling will result in the precipitation of insoluble chromium byproducts

which are very difficult to remove.

7 No purification is required. Very small amounts of chrysene may be observed via

NMR, though these will not react in subsequent steps and thus will be eliminated.

PAUSE POINT:

The product can be stored as a solid indefinitely.

Synthesis of 3,2-benzo[a]phenazine quinone

8 Dissolve 4.5 g (20 mmol) 2,3-dichloro-1,4-napthoquinone and 2.0 g (20 mmol) o-

phenylene diamine in 150 mL pyridine. This must be done by placing both materials into

the flask and then adding pyridine.

9 Attach a reflux condenser and bring the solution to reflux.

10 After 1 hour, allow the solution to cool to room temperature.

11 Filter the cooled solution. This should yield a dark brown-red solid. The solid can be

recrystallized from hot pyridine or carried on as is.

PAUSE POINT:

This intermediate can be stored for up to a week before further use.

Longer periods of storage may be possible.

12 After weighing the brown-red solid, place it in a second round bottom flask and add to

it 10 mL glacial acetic acid and 1 mL concentrated nitric acid. Subsequently add 0.66 mL

water per 1 g solid.

CAUTION:

Concentrated nitric acid is caustic and an oxidizer. Handle with care.

13 Heat the resultant solution in a boiling water bath for 1 hour.

CRITICAL STEP:

The reaction is complete when only yellow-orange precipitate

remains.

14 Filter this solution. The solid should be yellow.

15 Rinse the resulting solid with 15 mL ethanol and 15 mL diethyl ether.

CAUTION:

Do not let the washes contact the nitric acid solution. Use a new flask.

16 The final product can be purified by recrystallization from 7:3 chloroform:ethyl acetate,

but further purification is usually not necessary.

PAUSE POINT:

The product can be stored as a solid indefinitely.

Synthesis of [Rh(bpy)

]Cl

17 Dissolve 0.64 g rhodium chloride hydrate (2.8 mmol) and 50 mg hydrazine

monohydrochloride in 12.5 mL deionized water in a 50 mL round bottom flask.

18 Add a solution of 0.85 g (5.6 mmol) 2,2

′

-bipyridyl in 20 mL ethanol and de-oxygenate

the resultant solution by the repeated application of vacuum followed by backfilling with

Ar (g).

19 Bring the reaction mixture to reflux and heat until all materials have dissolved and

formed a yellow-orange solution.

20 While still hot, filter the reaction mixture through a medium glass frit.

CAUTION:

Hot-filtering, while necessary in this case, is always dangerous. Be careful.

21 Chill the filtrate overnight to promote crystallization.

Zeglis and Barton

Page 8

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

22 Isolate the yellow product crystals via filtration with a medium glass frit.

PAUSE POINT:

The product can be stored as a solid indefinitely.

Synthesis of [Rh(bpy)

(OTf)

]OTf

23 Add 500 mg Rh(bpy)

(Cl)

(1.0 mmol) to a 50 mL Schlenk flask with a 14/20 joint

on top and a side-arm and de-oxygenate the flask by evacuating it and refilling with Ar(g)

three times.

24 Carefully crack open the ampule of triflic acid and use a glass Pasteur pipette to add 5

g (excess) trflic acid to the reaction vessel under positive argon pressure.

CAUTION:

Triflic acid is very reactive and pyrophoric. Handle quickly and with care.

25 After adding the triflic acid, place a close the flask with a rubber septum, pierce the

septum with a needle, and purge the flask with argon for 30–60 seconds.

26 Allow the reaction mixture to stir for 16 h. Purge occasionally (every 4 to 6 h) to remove

HCl generated by the reaction.

27 After 16 h, cool 300 mL diethyl ether to

−

78° C in a dry ice/acetone bath. Place the

bath on a stir plate and add a stir bar to the flask.

28 Using a Pasteur pipette, add the reaction mixture drop-wise to the rapidly stirring, cold

diethyl ether. A yellowish-white powder will precipitate. No purification is necessary.

CRITICAL STEP:

It is imperative that the diethyl ether solution is stirring, so that each

new drop of reaction mixture falls in to cold diethyl ether and thus prompts product

precipitation.

PAUSE POINT:

While it is best to procedure directly to the next step, this product can

be stored for short periods of time in a dessicator.

Synthesis of [Rh(bpy)

(NH

)

](X)

(where X = PF

−

or OTf

−

)

29 Combine 500 mg Rh(bpy)

(OTf)

(0.6 mmol) and 20–50 mL concentrated NH

in a 250 mL round bottom flask fitted with a reflux condenser. The starting material should

be relatively insoluble in NH

OH.

30 Bring the mixture to reflux and boil it until all the material has gone into solution (5–

10 min).

31 Depending on the desired counter-ion, the product can be isolated using option A or

option B:

Precipitate from NH

OH solution with NH

Add excess NH

to the ammonia solution.

ii.

Chill overnight at 4°C to facilitate precipitation.

iii.

Filter with a medium frit to isolate the product.

Remove solvent under vacuum.

Remove all NH

OH with rotary evaporation. This will yield the

triflate salt of the product.

PAUSE POINT:

The product can be stored as a solid indefinitely.

Zeglis and Barton

Page 9

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Synthesis of [Rh(bpy)

(chrysi)](Cl)

32 In a 100 mL round bottom flask, dissolve 195 mg [Rh(bpy)

(NH

)

](X)

(approximately 0.2 mmol) and 57 mg 5,6-chrysene quinone (0.22 mmol) in 50 mL

acetonitrile with rapid stirring under ambient conditions.

33 Add 2 mL aqueous sodium hydroxide (0.4 M) and close the vessel to prevent

evaporation.

34 After 3 h, halt the reaction by bringing the pH of the solution to 7 by adding a

stoichiometric amount of HCl. By this point the reaction should have changed color

dramatically, from orange/yellow to dark red. Alternatively, one can monitor the reaction

by TLC using Silica F plates in a solvent system of 3:1:1 MeCN:H

O:MeOH with 0.1 M

KNO

35 Remove acetonitrile in vacuo by rotary evaporation.

36 Redissolve the reaction mixture in a minimum volume of water.

PAUSE POINT:

The reaction mixture can stand at room temperature while the ion

exchange column is being prepared.

37 Equilibrate 20 g of Sephadex SP-C25 ion exchange resin with MgCl

by making a

slurry of the resin in approximately 500 mL 0.05 M MgCl

38 With this equilibrated mixture, fill a 1–1.5 inch diameter column with about 5 inches

of resin.

39 Flush the excess MgCl

solution out of the column with 250 mL H

40 Flush the column with 500 mL deionized water.

41 Load the Rh(bpy)

(chrysi)

by pushing the diluted reaction mixture through the

column. The rhodium complex should stick to the uppermost layer of resin, creating a red

band at the top of the column.

42 Once all the reaction mixture has been loaded onto the column, wash the column with

250 mL deionized H

43 Elute the metal complex by slowly increasing the MgCl

concentration of the eluant

in 500 mL batches. Start with 0.01 M and increase by 0.01 M increments until 0.10 M

MgCl

; at this point, switch to 0.05 M increases until the metal complex (red band) has

been eluted (most likely around 0.3 M MgCl

CRITICAL STEP:

It is very important that the MgCl

concentration gradient is shallow.

Too steep a gradient will cause the ion exchange column to “crack”, leading to band

smearing and poor separation.

44 Concentrate the product-containing fractions on a reverse-phase cartridge (i.e. Waters

2 g C18 Sep-Pak Cartridge) primed with methanol.

45 Wash the cartridge thoroughly with water.

46 Elute the product with 1:1:0.001 H

O:MeCN:TFA.

47 Remove the solvent by rotary evaporation or lyophilization.

PAUSE POINT:

The finished product can be stored as a solid indefinitely.

NOTE:

representative UV-Visible spectrum of the product is shown in Fig. 7.

Zeglis and Barton

Page 10

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Synthesis of [Rh(bpy)

(phzi)](Cl)

48 In a 100 mL round bottom flask, dissolve 100 mg [Rh(bpy)

(NH

)

](X)

(approximately 0.1 mmol) and 35 mg 3,2-benzo[a]phenazine quinone (0.125 mmol) in 50

mL acetonitrile with rapid stirring under ambient conditions.

49 Add 2 mL aqueous sodium hydroxide (0.4 M) and close the vessel to prevent

evaporation.

50 After three h, halt the reaction by bringing the pH of the solution to 7 by adding water

and a stoichiometric amount of HCl.

51 Remove the acetonitrile in vacuo by rotary evaporation.

52 Redissolve the product in a minimum volume of water.

PAUSE POINT:

The reaction mixture can stand at room temperature while the ion

exchange column is being prepared.

53 Equilibrate 20 g of Sephadex SP-C25 ion exchange resin with MgCl

by making a

slurry of the resin in approximately 500 mL 0.05 M MgCl

54 With this equilibrated mixture, fill a 1–1.5 inch diameter column with about 5 inches

of resin.

55 Flush the excess MgCl

solution out of the column with 250 mL water.

56 Flush the column with 500 mL deionized water.

57 Load the Rh(bpy)

(phzi)

by pushing the diluted reaction mixture through the column.

The rhodium complex should stick, yielding a dark yellow/brown band at the top of the

ion exchange column.

58 Once all the reaction mixture has been loaded onto the column, wash the column with

250 mL deionized H

59 Elute the metal complex by slowly increasing the MgCl

concentration of the eluant

in 500 mL batches. Start with 0.01M and increase by 0.01M increments until 0.10M

MgCl

; at this point, switch to .05M increases until the metal complex (dark band) has

been eluted (most likely around 0.3 M MgCl

Critical Step:

It is very important that the MgCl

concentration gradient is shallow. Too

steep a gradient will cause the ion exchange column to “crack”, leading to band smearing

and poor separation.

60 Concentrate the product-containing fractions on a reverse phase cartridge (i.e. Waters

2 g C18 Sep-Pak Cartridge) primed with methanol.

61 Wash the cartridge thoroughly with water.

62 Elute the product with 1:1:0.001 water:MeCN:TFA.

63 Remove the solvent by rotary evaporation or lyophilization.

PAUSE POINT:

The finished product can be stored as a solid indefinitely.

NOTE:

A representative UV-Visible spectrum of the product is shown in Fig. 7.

Enantiomeric Separation of Rh(bpy)

(chrysi)

64 Fill one very long column (1.70 m × 1.5 cm) and two smaller columns (0.5 m × 1.5

cm, hereafter referred to as guard columns) with Sephadex

SP-C25 ion exchange resin

equilibrated with water.

Zeglis and Barton

Page 11

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

65 Prepare 4 L of a solution of 0.15 M (+)-KSb-tartrate in water. Filter this solution.

CAUTION:

(+)-KSb-tartrate is toxic. Handle with care and dispose of properly.

66 Elute all three columns thoroughly with the 0.15 M (+)-KSb-tartrate solution. Do not

recycle this eluant.

CRITICAL STEP:

Upon elution of the KSb-tartrate solution, the resin will compact a

little bit (20%); this may necessitate the addition of more resin to fill the columns all the

way.

68 Set aside on of the guard columns and set up the other two columns and eluant-recycling

pump as shown in Figure 8 . Make sure all three columns are full of both the chiral eluant

and ion exchange resin.

67 Dissolve 0.4 g

rac

-[Rh(bpy)

(chrysi)]Cl

in 5 mL water.

68 Carefully load the rhodium solution onto the top of the large column.

CRITICAL STEP:

To obtain good enantiomeric separation, it is very important that the

initial rhodium band on the column is very small. To ensure this, use a minimum of water

when first dissolving the rhodium complex and be careful when first loading the rhodium

solution onto the top of the large column.

69 Turn on the pump and allow the eluant to cycle. The separation will take about 2–3 d.

During cycling, monitor the column regularly (every 4 h during the day).

CRITICAL STEP:

Make sure all connections are secured with clamps or wire to prevent

leaks.

70 After about one day, separation should begin to become apparent. The first (lower,

faster) band is

-Rh(bpy)

(chrysi)

; the second (upper, slower) band is

-Rh

(bpy)

(chrysi)

. Detach the first guard column after it “catches” the lower band and

replace it with the second guard column. Cap the first guard column to prevent it from

drying.

CRITICAL STEP:

Do not let the guard columns or the main column dry out during the

separation. This will cause the compound to stick permanently to the resin.

71 Continue eluting the column (using the pump) until the second guard column fully

catches the second band.

72 After the second guard column has fully caught the second band, detach and cap it to

prevent any evaporation.

CAUTION:

All of the eluant and resin used will contain antimony. Elute the large column

with water and dispose of the antimony-containing eluant appropriately. Likewise, the

resin will be contaminated with antimony and should be disposed of properly.

73 Wash the two guard columns with 0.05 M MgCl

solution to remove the remaining

antimony tartrate.

CAUTION:

Be sure to dispose of the antimony-containing eluant properly.

CRITICAL STEP:

Be careful to keep all columns and product-containing eluants well-

labeled and separate during product isolation to prevent inadvertent re-racemization.

74 Remove the compound from the guard columns by washing the columns with 0.5 M

MgCl

until all the compound has been eluated.

75 Concentrate the two product fractions on 5 g Waters Sep-Pak C18 cartridges previously

primed with 2 × 10 mL methanol and 1 × 10 mL water.

Zeglis and Barton

Page 12

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

76 Wash the cartridges with 200 mL water.

77 Elute the products with a mixture of 1:1:0.001 MeCN:H

O:TFA.

78 Freeze the eluants with liquid N

and lyophilize to dryness. Due to some permanent

product adhesion to the ion exchange resin, only about 80% mass yield can be expected

from the separation. The two enantiomers should be obtained in about 1:1 ratio.

NOTE:

Representative circular dichroism spectra of both the

- and

-enantiomers are shown in

Figure 9.

Mismatch detection via photocleavage with 5

′

P]-labeled DNA and denaturing

PAGE

79 Make a 100

M stock solution of the single stranded DNA sequences (forward and

complement) to be investigated.

80 Label the 5

′

end of each of the strands with [

P]-ATP: combine 15

L water, 2

polynucleotide kinase buffer (provided), 1

L ssDNA stock solution, 1

L polynucleotide

kinase solution, and 1

P-ATP solution in a 1.7

L microcentrifuge tube. Vortex and

spin the reaction mixture in a microcentrifuge.

CAUTION:

Be sure to follow all standard radioactivity safety procedures to limit your

exposure.

CRITICAL STEP:

The mismatch-specific metallointercalators –Rh(bpy)

(chrysi)

, in

this case – will only cleave one strand of the mismatched DNA. Therefore, cleavage will

only be evident using one of the two labeled strands. While the location of cleavage has

been determined for most sequence contexts,

it is best to label both and determine

cleavage on both strands.

81 Incubate the labeling reaction for 2 h at 37°C.

82 After 2 h, add 80

L water, vortex, and purify using a Micro Bio-Spin® 6

Chromatography Column.

CAUTION:

Do not centrifuge the spin columns at greater than 3000 rpm; they will break.

83 Dry the samples on a lyophilizer or vacuum centrifuge.

84 Purify the 5

′

-labeled oligonucleotides via 20% PAGE:

Pour a 20% polyacrylamide gel.

Take up the dried oligonucleotide samples in 10

L denaturing formamide loading dye.

Load the sample onto the gel, and run the gel for 60–90 min at 90 Watts using 1X TBE

as the running buffer.

Remove one gel plate, leaving the gel affixed to the other gel plate.

Visualize the gel via X-ray

Cut out the parts of the gel that correspond to full-length, labeled DNA with a clean

razor blade.

With tweezers, place these gel bits into a clean centrifuge tube.

Add 1 mL 100 mM triethylammonium acetate pH 7.0 to each centrifuge tube containing

gel.

Incubate at 37°C overnight.

Remove triethylammonium solution and place in a clean 1.7 mL centrifuge tube.

Zeglis and Barton

Page 13

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Speedvac to remove solvent.

Take up dried sample in 100

L water and purify with a Micro Bio-Spin® 6

Chromatography Column.

Dry the samples on a vacuum centrifuge.

Take up labeled oligonucleotide in 50

L of 10 mM NaPi buffer, pH 7.1.

PAUSE POINT:

Labeled DNA can be stored for weeks at 4°C. However, it is best to

perform the experiments before the label decays too much.

85 Prepare an aqueous buffer solution of 100 mM NaCl and 20 mM NaPi, pH 7.1

86 Make a 2

M duplex stock solution for the irradiation experiments: combine 2

L of

each unlabeled DNA single strand (forward and complement), 4

L of one of the two

labeled oligonucleotide solutions, and 92

L 100 mM NaCl/20 mM NaPi buffer.

87 Anneal the 2

M duplex stock solution via heating to 90°C followed by gradual cooling.

CRITICAL STEP:

It is important that the solution cool to room temperature slowly to

ensure that proper hybridization has taken place. Our suggested method is placing the

centrifuge tube containing the solution on a 90°C heat block for 5 min, removing the entire

heat block, and allowing the block and sample tube to cool to room temperature together

over the course of about 90 min.

88 Prepare a stock of 2

M Rh(bpy)

(chrysi)

in water.

89 Prepare irradiation samples: in 1.7 mL centrifuge tubes, combine 10

L DNA stock

solution (with either forward or reverse strand 5

′

-labeled) and 10

L rhodium stock

solution to create a final 20

L sample containing 1

M DNA (with radiolabel) and 1

Rh(bpy)

(chrysi)

in a buffer of 50 mM NaCl and 10 mM NaPi, pH 7.1.

NOTE:

In addition to samples containing both DNA and Rh(bpy)

(chrysi)

, it is also

important to make control samples under various conditions: for example, DNA and

intercalator in the absence of irradiation, DNA alone in the presence of light, and DNA

alone in the absence of light.

90 Irradiate the samples (for more information see

EQUIPMENT SET-UP

91 Dry all samples under vacuum.

92 Determine the counts per minute (cpm) for each sample using a scintillation counter.

93 Dissolve each sample in enough denaturing formamide dye such that there are 10,000

cpm/

L (e.g. dissolve a sample containing 100,000 counts in 10

L loading dye).

PAUSE POINT:

Samples dissolved in denaturing formamide dye can be stored at room

temperature for days.

94 Load 5

L of each sample onto a 20% denaturing polyacrylamide gel.

95 Run the gel at 90 Watts for approximately 1 hour (or the amount of time it takes the

parent band, estimated using the running dyes, to travel about 2/5 of the way down the

plate).

96 Separate the gel plates, being careful that gel stays on just one of the plates.

97 Remove the gel from the plate using a large piece of film.

98 Cover the gel with Saran Wrap

99 Place the gel in a blanked phosphoroimager screen and expose it for 4 h.

Zeglis and Barton

Page 14

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

100 After 4 h, remove the gel from phosphorimager screen and develop it on a

phosphorimager.

101 Analyze the gel as a *.gel file in the ImageQuant program (Molecular Dynamics) or

as a *.tiff file in Photoshop (Adobe).

NOTE:

See Figure 10 for the result of a typical

PAGE experiment.

SNP detection with Rh(bpy)

(phzi)

NOTE:

For the sake of simplicity, the following protocol is specific to the synthetic

plasmid described earlier

. The procedure, however, can be easily adapted to any

biologically derived gene. For example, the paper mentioned above also discusses the

application of the procedure to SNP detection in the tumor necrosis factor (TNF)

promoter region. For a schematic of the technique described below, see Figure 10.

102 Amplify both genes in question, one known to have the correct sequence and one

suspected to contain a SNP, by PCR using primers containing restriction enzyme sites (in

this case, sites for

Cla

I and

Xho

I).

103 Add 6 units of

Exo

I and 20 units of calf alkaline phosphatase to each PCR reaction

and incubate for 1 h at 37°C to degrade excess primers and dNTPs.

104 To determine the success and purity of the PCR reaction, run out a small amount of

both PCR products on a 2% agarose gel pre-stained with ethidium bromide. Only a single

band should be present. The purity of this initial PCR is very important to the success of

the overall assay, so optimization may be required if the reaction appears messy or frequent

stops are visible.

105 Purify both PCR products by using a QIAquick PCR purification column eluting with

10 mM Tris•HCl (pH 8.5).

PAUSE POINT:

At this point, the PCR products can be stored for up to a month at 4°C.

106 Thermally denature the DNA under these low ionic strength conditions by heating to

99 °C for 30 min followed by immediate and rapid cooling to 4°C.

107 Combine equimolar concentrations of each PCR product in a buffer of 60 mM

Tris•HCl (pH 7.5), 10 mM MgCl

, 100 mM NaCl, and 1 mM dithioerythritol.

108 Re-anneal the pooled sample by heating to 95°C for 10 min and linearly decreasing

the temperature to 4°C over the course of 150 min. This procedure generates the

heterozygous duplexes that contain the photocleavable mismatch.

109 Add 5 units

Cla

I and 5 units

Xho

I to the pooled sample and incubate the duplex with

the restriction enzymes for 1 hour at 37°C followed by denaturing the enzymes at 80 °C

for 20 min.

110 Prepare a sample of 400 nM Rh(bpy)

(phzi)

in water.

111 In a 1.7 mL centrifuge tube, combine 10

L pooled DNA stock and 10

L Rh

(bpy)

(phzi)

stock to yield a final reaction mixture containing 200 nM Rh

(bpy)

(phzi)

, 30 mM Tris•HCl (pH 7.5), 5 mM MgCl

, 50 mM NaCl, and 0.5 mM

dithioerythritol.

112 Irradiate the samples (for more information on irradiation see

EQUIPMENT

SETUP

113 Dry the samples in vacuo.

PAUSE POINT:

Dried samples can be left overnight at room temperature or for a few

days at 4°C.

Zeglis and Barton

Page 15

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

114 Add fluorescent tags to the strands by single base extension using the Applied

Biosystems SNaPshot

kit. The two strands, forward and reverse, will receive different

fluorescent tags, resulting in two different “color” signals on the capillary electrophoresis

trace, one “color” for the forward strand and a different “color” for the reverse strand.

115 Add 2 units calf intestinal alkaline phosphatase to each sample and incubate 1 hour

at 37°C followed by denaturing the enzyme at 80°C for 20 min.

116 Ethanol precipitate each sample and remove left-over solvent in vacuo.

117 Re-dissolve each sample in 1

L water and 1

L molecular weight standard (see

EQUIPMENT SETUP

for more information on the molecular weight standard).

118 Add 24

L deionized formamide to each sample and heat to 95°C for 5 min.

119 Cool the samples to 4°C for 5 min.

120 Load samples on an ABI Prism 310 capillary electrophoresis instrument and analyze

results. For more information on the configuration of the ABI instrument, see

EQUIPMENT SETUP

NOTE:

Sample capillary electrophoresis traces for assayed wildtype and SNP-containing

genes are shown in Figure 12.

TIMING

Synthesis of 5,6-chrysene quinone:

Steps 1–3, 24 h; Steps 4–7, 45 min. Total: 24.75 h.

Synthesis of 3,2-benzo[a]phenazine quinone:

Steps 8–10, 90 min.; 11–16, 2 h. Total: 3.5 h.

Synthesis of [Rh(bpy)

]Cl:

Steps 17–18, 30 min.; Steps 19–20, 45 min.; Steps 21–22, 14

h. Total: 15.25 h.

Synthesis of [Rh(bpy)

(OTf)

]OTf:

Step 23, 10 min.; Step 24–26, 16 h; Step 27–28, 1 h.

Total: 17 h.

Synthesis of [Rh(bpy)

(NH

)

](X)

: Step 29–30, 15 min.; Steps 31Ai–31Aiii, 16 h; Step 31B,

45 min. Total: 1 h or 16.25 h depending on Step 31.

Synthesis of [Rh(bpy)

(chrysi)](Cl)

: Steps 32–35, 3 h; Steps 36–43, 5 h; Steps 44–46, 10

min.; Step 47, 12 h. Total: 20 h.

Synthesis of [Rh(bpy)

(phzi)](Cl)

: Steps 48–51, 3h; Steps 52–59, 5h; Steps 60–62, 10 min.;

Step 63, 12 h. Total: 20 h.

Enantiomeric Separation of Rh(bpy)

(chrysi)

: Steps 64–68, 3 h; Steps 69–71, 3 d; Steps

73–78, 3 h. Total: 3.5 d.

Mismatch detection with Rh(bpy)

(chrysi)

analyzed by PAGE:

Step 79, 10 min.; Steps

80–83, 2.5 h; Steps 84, 1 d; Steps 85–86, 30 min.; Step 87, 1.5 h; Steps 88–90, 1.5 h; Step 91,

1 h; Steps 92–93, 30 min.; Steps 94–95, 1.5 h; Steps 96–101, 5 h. Total: 3 d.

SNP detection with Rh(bpy)

(phzi)

analyzed by capillary electrophoresis:

Step 102, 4

h; Step 103, 1 h; Step 104, 3 h; Step 105, 30 min.; Step 106, 30 min.; Step 107–108, 3 h; step

109, 1 h; Step 110–113, 2 h; Step 114, 30 min.; Step 115, 1.5 h; Step 116–119, 30 min.; Step

120, 2 h. Total: 3 d.

Zeglis and Barton

Page 16

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

TROUBLE-SHOOTING

For troubleshooting advice, see Table 1.

ANTICIPATED RESULTS

Typical Synthetic Yields

5,6-chrysene quinone (85%); 3,2-benzo[a]phenazine quinone (60%); [Rh(bpy)

(Cl)

]Cl

(60%); [Rh(bpy)

(OTf)

]OTf (30–40%); [Rh(bpy)

(NH

)

](X)

(30% via precipitation, 95%

via rotary evaporation); [Rh(bpy)

(chrysi)](Cl)

(80%); [Rh(bpy)

(phzi)](Cl)

(50%).

Product Appearance

chrysene quinone, orange powder; 3,2-benzo[a]phenazine quinone intermediate, red powder;

3,2-benzo[a]phenazine quinone, yellow solid; [Rh(bpy)

]Cl, yellow crystalline solid; [Rh

(bpy)

(OTf)

]OTf, yellow-white powder; [Rh(bpy)

(NH

)

](X)

, white powder; [Rh

(bpy)

(chrysi)](Cl)

, brownish-red powder; [Rh(bpy)

(phzi)](Cl)

, brownish-yellow powder;

-[Rh(bpy)

(chrysi)](Cl)

, browinish-red powder;

-[Rh(bpy)

(chrysi)](Cl)

, browinish-red

powder.

Analytical Data

5,6-Chrysene quinone:

H NMR (CD

)

9.4 (d, 1H), 8.85 (d, 1H), 8.8 (d, 1H), 8.5–7.5

(m, 7H); MS (m/z): 259 (M+H

3,2-Benzo[a]phenazine quinone:

H NMR (CD

)

8.8 (d, 1H), 8.3–8.1 (m, 3H), 8.1–7.8

(m, 3H), 7.7 (t, 1H); MS (m/z): 261 (M+H

[Rh(bpy)

(Cl)

]Cl:

H NMR (d

-DMSO):

9.71 (d, 2H), 9.01 (d, 2H), 8.90 (d, 2H), 8.63 (t,

2H), 8.33 (t, 2H), 8.17 (t, 2H), 7.82 (d. 2H), 7.59 (t, 2H); FAB-MS (m/z): 485.96.

[Rh(bpy)

(OTf)

]OTf:

H NMR (d

-DMSO):

9.17 (d, 2H), 9.08 (d, 2H), 8.90 (d, 2H), 8.80

(t, 2H), 8.4 (m, 4H), 7.8 (d, 2H), 7.7 (t. 2H); FAB-MS (m/z): 712.9.

[Rh(bpy)

(NH

)

](X)

H NMR (d

-acetone):

9.44 (d, 2H), 9.05 (d, 2H), 8.89 (d, 2H), 8.75

(t, 2H), 8.45 (t, 2H), 8.30 (t, 2H), 8.03 (d. 2H), 7.75 (t, 2H), 5.06 (broad singlet, 6H); FAB-MS

(m/z): 449.

[Rh(bpy)

(chrysi)](Cl)

H NMR (d4-methanol):

8.94 (t, 2H), 8.86 (t, 2H), 8.80 (d, 1H),

8.77 (d, 1H), 8.56 (split t, 2H), 8.44 (m, 5H), 8.4 (d, 1H), 8.15 (m, 1H), 8.03 (m, 1H), 7.95 (m,

3H), 7.86 (d, 1H), 7.81 (d, 1H), 7.64 (m, 5H); FAB-MS (m/z): 671; UV-Vis (Figure 7):

max

303 nm (

=57,000 M

−

), 315 nm (

=52,200 M

−

), 391 nm (

=10,600 M

−

-[Rh(bpy)

(chrysi)](Cl)

: Circular Dichroism (H

O, see Figure 9): 233 (34), 264 (26), 286

(

−

12), 308 (

−

42), 318 (

−

100), 341 (6).

-[Rh(bpy)

(chrysi)](Cl)

: Circular Dichroism (H

O, see Figure 9): 233 (

−

34), 264 (

−

26),

286 (12), 308 (42), 318 (100), 341 (

−

6).

[Rh(bpy)

(phzi)](Cl)

H NMR (d

-DMSO):

14.88 (s, 1H), 14.70 (s, 1H), 9.03 (m, 4H),

8.90 (d, 2H), 8.72 (d, 1H), 8.6 (t, 2H), 8.54 (d, 1H), 8.47 (t, 2H), 8.32 (d, 1H), 8.2 (d, 1H), 8.11

(t, 1H), 8.03 (m, 3H), 7.94 (t, 1H), 7.84 (t, 1H), 7.75 (t, 3H), 7.69 (d, 1H); ESI-MS (m/z): 671

(m-2H

); UV-Vis (Figure 7):

max

: 304 nm (

=65,800 M

−

), 314 nm (

=67,300 M

−

), 343 nm

(

=39,300 M

−

Zeglis and Barton

Page 17

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Acknowledgments

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

support. Additionally we are grateful to our coworkers, J. Hart, I. Lau, V. Pierre, and R. Ernst, for their help in working

out experimental details.

References

1. Erkkila KE, Odom DT, Barton JK. Recognition and reaction of metallointercalators with DNA. Chem

Rev 1999;99:2777–2795. [PubMed: 11749500]

2. Kielkopf CL, Erkkila KE, Hudson BP, Barton JK, Rees DC. Structure of a photoactive rhodium

complex intercalated into DNA. Nat Struct Bio 2000;7:117–121. [PubMed: 10655613]

3. Jackson BA, Barton JK. Recognition of DNA base mismatches by a rhodium intercalator. J Am Chem

Soc 1997;119:12986–12987.

4. Jackson BA, Alekseyev VY, Barton JK. A versatile mismatch recognition agent: specific cleavage of

a plasmid DNA at a single base mispair. Biochem 1999;38:4655–4662. [PubMed: 10200152]

5. Jackson BA, Barton JK. Recognition of base mismatches in DNA by 5,6-chrysenequinone diimine

complexes of rhodium(III): a proposed mechanism for preferential binding in destabilized regions of

the double helix. Biochem 2000;39:6176–6182. [PubMed: 10821692]

6. Modrich P. Mechanisms and biological effects of mismatch repair. Ann Rev Genet 1991;25:229–253.

[PubMed: 1812808]

7. Kolodner R. Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev 1996;10:1433–

1442. [PubMed: 8666228]

8. Harfe BD, Jinks-Robertson S. DNA mismatch repair and genetic instability. Ann Rev Genet

2000;34:359–399. [PubMed: 11092832]

9. Modrich P.

Mechanisms in eukaryotic mismatch repair. Biol Chem 2006;41:30305–30309.

10. Kolodner RD. Mismatch repair: mechanisms and relationship to cancer susceptibility. Trends

Biochem Sci 1995;20:397–401. [PubMed: 8533151]

11. Arzimanoglou II, Gilbert F, Barber HRK. Microsatellite instability in human solid tumors. Cancer

1998;82:1808–1820. [PubMed: 9587112]

12. Loeb LA, Loeb KR, Anderson JP. Multiple mutations and cancer. Proc Natl Acad Sci 2003;100:776–

781. [PubMed: 12552134]

13. Junicke H, Hart JR, Kisko J, Glebov O, Kirsch IR, Barton JK. A rhodium(III) complex for high-

affinity DNA base-pair mismatch recognition. Proc Nat Acad Sci 2003;100:3737–3742. [PubMed:

12610209]

14. Syvänen A. Acessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev

2001;2:930–942.

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

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

16. Mürner H, Jackson BA, Barton JK. A versatile synthetic approach to rhodium(III) diimine

intercalators: condensation of

ortho

-quinones with coordinated

cis

-ammines. Inorg Chem

1998;37:3007–3012.

17. Krotz AH, Kuo LY, Barton JK. Metallointercalators: syntheses, structures, and photochemical

characterizations of phenanthrenequinone diimine complexes of rhodium(III). Inorg Chem

1993;32:5963–5974.

18. Ferrari M, Carrera P, Cremonesi L. Different approaches to molecular scanning of point mutations

in genetic diseases. Pure Appl Chem 1996;68:1913–1918.

19. Kwok PY, Deng Q, Zakeri H, Taylor AL, Nickerson DA. Increasing the information content of STS-

based genome maps: identifying polymorphisms in mapped STSs. Genomics 1996;31:123–126.

[PubMed: 8808290]

20. Taillon-Miller P, Piernot EE, Kwok PY. Efficient approach to unique single nucleotide polymorphism

discovery. Genome Res 1999;9:499–505. [PubMed: 10330130]

21. Zhou W. Mapping genetic alterations in tumors with single nucleotide polymorphisms. Curr Opin

Onc 2003;15:50–54.

Zeglis and Barton

Page 18

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

22. Schork NJ, Fallin D, Lanchbury JS. Single nucleotide polymorphisms and the future of genetic

epidemiology. Clinical Gentics 2000;58:250–264.

23. Rider MJ, Tobe VT, Taylor SL, Nickerson DA. Automating the identification of DNA variations

using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome.

Nucleic Acids Res 1998;26:967–973. [PubMed: 9461455]

24. Petitjean A, Barton JK. Tuning the DNA-reactivity of cisplatin: conjugatin to a mismatch-specific

metallointercalator. J Am Chem Soc 2004;126:14728–14729. [PubMed: 15535691]

25. Schatzschneider U, Barton JK. Bifunctional rhodium intercalator conjugates as mismatch-directing

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

26. Zeglis BM, Barton JK. A mismatch-selective bifunctional rhodium-oregon green conjugate: a

fluorescent probe for mismatched DNA. J Am Chem Soc 2006;128:5654–5655. [PubMed:

16637630]

27. Brunner J, Barton JK. Targeting DNA mismatches with rhodium intercalators functionalized with a

cell-penetrating peptide. Biochem 2006;45:12295–12302. [PubMed: 17014082]

28. Hart JR, Glebov O, Ernst RJ, Kirsch IR, Barton JK. DNA mismatch-specific targeting and

hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators. Proc Nat Acad

Sci 2006;103:15359–15363. [PubMed: 17030786]

Zeglis and Barton

Page 19

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Figure 1.

Schematic of the use of mismatch-selective metallointercalators in detection of both single

nucleotide polymorphisms (SNPs) (top) and single base mismatches (bottom).

Zeglis and Barton

Page 20

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Figure 2.

-[Rh(bpy)

(chrysi)]

(left) and

-[Rh(bpy)

(phzi)]

(right). In some cases (ref.

, for

example), these complexes are referred to as Rh(chrysi) and Rh(phzi).

Zeglis and Barton

Page 21

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript

Figure 3.

Structural model of Rh(bpy)

(chrysi)

bound to a CA mismatch (adapted from Pierre, Kaiser,

Barton, submitted). The bulky metal complex (red) inserts into the DNA base stack (gray) and

the mismatched bases (blue) are extruded.

Zeglis and Barton

Page 22

Nat Protoc

. Author manuscript; available in PMC 2009 September 30.

NIH-PA Author Manuscript