Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing

BIOCHEMICAL

Vol. 169, No. 1, 1990

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS Pages 87-94

May 31, 1990

PRBFGRRNTIAL

IIODIFICATION

DTMRTHYLDLOXIRANE

R.

Jeremy

AND

H.

Division

of

D.

16,

R.

and

of

Boyd’,

of

Chemistry, BT9

TO

DNA

DNA

BY

SlZQURNCclNG

Kumarl,

Stevenson’

Biology

and

Queen’s

78L,

IN

Shiv

Clarke

School

School

8A3BS

APPLICATION

Sharma’

Belfast

March

CDANINB

Derek

Biochemistry, 2

and

Received

IT3

Davies’,

Rarain 1

OF

Biochemistry,

University,

Northern

Treland

1990

32 Susasary: From gel sequencing experiments with P-end-labelled oligodeoxyribonucleotides. it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3’or S.-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA. 01990 Academic Press, Inc.

0 Dimetbyldioxirane powerful

(CH3j2

oxidising

a wide

range

agent

which

of

organic

conditions

(1.2).

It

compounds,

hydroxylamines

or

Product

isolation

nitrones

(3),

dioxirane the

and

biochemistry

(4-7).

of

is

modified DNA

by with

predominantly

to

map

DNA the

is

for

capable

is

relation

to

to

information

the

dimethyldioxirane

produces

position

sites

of

piperidine of

guanine causes

guanine

Dimethyldioxirane foxone) to at room temperature,

DNA

has of

this

bases. selective in

AND

sequencing

Although studied

radiation

and

its we

unknown

(81,

components

show

that

are treatment

structure) heating

cleavage

of which

the can

be

used

experiments.

METHODS

was prepared by adding potassium a mixture of acetone and aqueous as described by Nurray and

87

into

dimethyl-

ionising

Subsequent chain

into

nitro

volatile. extensively

DNA

(of

to imines

both

communication, lesions

atom reaction

amines

highly

how

oxygen

converts

been

effects

residues

MTRRTALS klaterials: monoperoxysulphate bicarbonate,

are

concerning In

the

and because

acetone

extremely

neutral

oxidise (2)

simplified

damage

an

mild.

cleanly

&oxides

and

inserting

under

example, amine

versatile,

of

groups

dimethyldioxirane.

with

a new,

by-product

in no

at

modified

can,

oxidative

currently

is

functional

reaction

particularly

there

of

its

C
sodium Jeyaraman

(9).

0006-291x/90 $1.50 Copyright 0 1990 by Academic Press. kc. All rights of reproduction in any form reserved

Vol.

169, No. 1, 1990

It was isolated vessel into a temperature. was determined dimethyldioxirane mixed with a liberated is stock solutions

in

required

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

as a solution in acetone by distillation from the generation series of cold traps (9) maintained at dry ice-acetone The concentration of dimethyldioxirane in this stock solution by iodometrfc titration (10). In this procedure, the solution is diluted with 60% acetic acid in acetone and saturated aqueous solution of potassium iodide; the iodine titrated with standardized sodium thiosulphate. In practice, of dimethyldioxirane in acetone were found to have

concentrations as

BIOCHEMICAL

with

the

range

a 1:l

SO-100

They

s#f.

acetone/water

were

mixture

stored

at

at

-7OOC

and

tbe

elution

[o-32PIddATP terminal Amersbam

by

(110 TBq deoxynucleotidyl International.

grid-labelled

technique

aaaol-1

was

lebelled

kinase and GTAGGGGCCAC-3’ by the Double annealing sequence.

32

with

[r-32PlATP was

also

(12).

), T4 transferase

oligonucleotide~:

TCC-3’

P at

polynucleotide (calf

[y-32PlATP

buffer,

Treatment solutions adenosine, tbymidine

of en

mmol-‘),

(E.coli obtained

B),

end from

its

5’-end

by

45-mar at its

action

to

of

T4

polynucleotide

S’-GMGGCTGGCATGGCGCTCTGTGTGGGCTGChCAS.-end by this method or at

92OC and by ethanol

its

3*-end

[u- 32PlddATP and were prepared by of complementary dissolved in 100 mX

allowed to precipitation

cool

with dimetbyldioxirane: each of the deoxyribonucleosides ?‘-deoxycytidine, 5-methyl-2’-deoxycytidine equirnolar quantity of dlmetbyldioxirene

slowly (11).

(14).

to

room

Aqueous 2’-deoxyand Pl,

(110

in

acetone) in Bppendorf vials and maintained at 22OC for 1 b; the mixtures were then lyopbilised (Savant Speedvec) and reconstituted (SO0 ~1). To determine the extent of reaction, the samples were by reversed-phase HPLC on a u Bondapak Cl8 column (300 x 4 aan) with

equipment

described elsewhere (15). After sample injection, the column -1 eluted, at a flow rate of 0.8 ml min , with 0.05% aqueous trifluoroacet3c acid for 5 min followed by a linear gradient to 0.05% trifluoroacetic containing 30% methanol after 15 min; elutfon profiles were monitored 254 ma. In each case, the peak corresponding to the unchanged deoxyribonucleoside starting material was collected end tben quantified spectropbotometry.

Gel

an

by recovered

5’-CCCAGThGATTCGTGACMAGCCGCATAhCCthe

the

with

34-mer

was heated was recovered

of 2’-deoxvribonucleosides (500 Pl, 20 m#T) ?‘-deoxyguanosine, were mixed with

TBq

Sigma;

The

The (13). labelled

pH 8.0, duplex

tbe

(110

kinese were

thymus)

action of terminal deoxynucleotidyl transferase stranded molecules of the end-labelled 34-mer it with en unlabelled synthetic oligonucleotide An equimolar mixture of the two strands

Tris-HCl temperature;

90 mD reaction water analysed

diffusion

diluted

O°C.

Z’Deoxyribonucleosides and calf thymus DNA were obtained frora DNA was sheared and deproteinised before use (11). Synthetic oligodeoxyribonucleotides were assembled following standard procedures Applied Biosystems 380 B DNA Synthesfser using 8-cyanoethyl-7#,!-diisopropylamino-phosphoramidite reagents; after deprotection they were purified electrophoresis on 20% polyacrylamide gels containing 7 W urea and by

then

secruencing

end cleavage, sequencing 7 W urea. Macropbor autoradiography

of 32 gels

olironucleotides: P-end-labelled (40 x 18

Blectropboresis gel electrophoresis at

-7OOC

After oligonucleotides 0.3 lplll thick)

cm, was (Fuji

performed, system; X-ray

the film).

88

base-specific were containing

chemical

on

was acid at by

in!

modification

fractionated 16% polyacryluide

at 45’C end 25 W, gels were subsequently

in

an

on and IJCB visualised

by

Vol.

169,

No.

1, 1990

Ease-specific Raxam and Gilbert DNA at A + G.

BIOCHEMICAL

cleavages (13); the

AND

BIOPHYSICAL

RESEARCH

at G, T, and C + T were carried out according to method of Banasxuk et al. (16) was used to cleave

Action of dimethyldioxirane on svnthetic oligonucleotides and dimethyldioxirane

oligonucleotides: were carried out

Rppendorf dissolved mg ml -1 ).

vials. The 32 P-end-labelled in water (5 vl) and mixed

oligonucleotide (20-40 1 ul of aqueous calf

O°C while

freshly

After

The solution

mixing,

before chilled freshly

the

with

of oligonucleotide

diluted

COMMUNICATIONS

plus

dimethyldioxirane

reaction

vial

carrier

(20 ul;

was held

between 1.5 ml

pmol) was thymus DIU (4

DNA was maintained 0.1

in a water

Reactions siliconised

in

at

- 2 mM) was added.

bath

at 20°C

for

2 min

being placed in another water bath at 85OC for a further 5 min. The contents of the vial were then lyophilised prior to treatment with diluted 1 M piperidine (40 Pl) which was carried out by heating in

sealed glass capillary tubes at 90°C for 30 min. After chilling in ice, the piperidine solutions were transferred to new Eppendorf vials, dried, and then lyophilised three times from 30 ul of water. The pellets were dissolved in 80% formamide loading solution (11) and loaded onto a denaturing polyacrylamide sequencing gel which was run under the conditions described above.

RESULTS MID DISCUSSION In

preliminary

experiments

2'-deoxyribonucleoside

components

Bach

assessed.

starting

(dT). which

at material

that lack

respectively W-absorbing

of DR.4 towards

(80%) of

occurred the

with

a conjugated and the products

deoxyadenosine

(d.A).

with

chromophore.

reaction,

However, the W spectrum new peaks.. limited conversion to the g(l)-oxide

the

1 (c)

of of

(dG)

non-W extent

had occurred,

There

was now total

degradation the

with

individual

of

the

2 mol

of dimethyldioxirane

destruction others.

shown)

this

deoxyribonucleosides

of has

investigation. 89

not

the

oxidation yet

of

revealed

no

that

substance

co-eluting

of the is in the order the experiments

of deoxyribonucleoside.

of dG and dT and a proportionate The nature

some

dA indicated

per mol

and

(< 5%) destruction

Based on these results, the susceptibility towards oxidation by dimethyldioxirane when dG - dT > dC - m5dC > d.4. This trend was confirmed repeated

(dC)

show that (not

recovered

species

of 30% and 20%

little

with dh on HPLC. deoxyribonucleosides were

and (b))

absorbing

HPLC profile the

and thymidine

1 (a)

and (d))

was very

its of

quantity

Deoxycytidine

to

(Fig. There

was

amount of unchanged Rxtensive destruction

(Fig.

into

were degraded

HPLC profiles are formed. After

products mainly

the

an equimolar

the

deoxyguanosine

reaction

r-electron (m'dC>

of

dimethyldioxirane

was treated

dG and dT are converted

S-methyldeoxycytidine

reactivity

22OC for 1 h following which was estimated by HPLC analysis.

The HPLC profiles

indicate

relative

deoryribonucleoside

dimethyldioxirane deoxyribonucleoside the

the

products

been determined

increase

in

derived

from

but

is under

Vol.

BIOCHEMICAL

169, No. 1, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(bl

1.5

$

dc

10

2

0.5

m5dC

(Cl

Cd1

_A-

I 0

cA 20 0 Ehtion Tie (mid

b

10

20

Fir. 1. HPLC elution profiles (at 254 nm) of 2'-deoxyrihonucleosides with so equimolsr quantity of dimethyldioxirene: (a) deoxyguanosine (b) thy&dine (dT). (c) deoxycytidiae (dC). (d) S-methyldeoxycytidiae Details of the HPLC system sre given under Hethods.

The modification explored

by gel

of

nucleotide

sequencing

residues

experiments

in DNA by dimethyldioxirane 32 P-end-labelled synthetic

witb

An approximately

oligonucleotides containing 34 or 45 bases. of carrier DNA was mixed with the labelled with mol

dimetbyldioxirane. per mol

allowed

of

An amount

total

to react

nucleotide

for

2 min

residual

dimetbyldioxirane.

presence

of frank

gels

with

reactions.

reference

chain

of

before

to fragments

or base produced

in the

was added beat3ng

Tbe oligonucleotides breaks

before

dimetbyldioxirane,

labile

to

each

to SS°C to were

cleavage

by conventional

was

lo-fold

oligonucleotide

phosphate,

at 20°C

treated (dC), (a'dc).

tben sites

excess treating

range

sample

0.3

it to

and

decompose analysed

for

the

on DM~ sequencing chemical

sequencing

3

Vol.

169, No. 1, 1990

As

evidenced

in

dimethyldioxirane scission

Fig.

under of

DNA

with

the

fragments

the

BIOCHEMICAL

AND BIOPHYSICAL

2

and

(lanes

the

10

conditions

caused

produced

standard

guanine-specific

obtained

far

both

1

did

backbone. selective

having

the

single

C A ?CdG 2 3 4 5

at

same mobility

the

not

stranded

DNA

cause

appreciable

of

the

results (Fig.

treated

C;uanine

generated

Similar 34-mer

to

heating

sites

as those

procedure.

and double

of

However,

cleavage

Uaxam-Gilbert

the

exposure

employed

deoxyrlbose-phosphate

piperidine

151,

RESEARCH COMMUNICATIONS

bases,

by the were

2, lanes

7 to

G 6

7 6

9

10

11 12

13

14

15 16

FiK. 2. Autorsdiogropb of [5S-32Pl end-lsbelled single and double stranded 34-mer treated with dimetbyldiozirane and plperidine. Lanes 1 sod 2: uncut 34-mer before and after besting witb piperfdine; lanes 3 to 6 and 16: products of HaxamGilbert sequencing reactions (13) ss indicated; lanes 7 to 9: single stranded 34-mer (20 plol) plus carrier DNA (4 ug) reacted witb 7, 14 and 22 nmol of dimetbyldioxirane respectively then heated with piperidine; lanes 11 (20 ~1) plus carrier iMA (4 vg) reacted with to 14: double stranded 34-mer 7, 14, 22 end 36 nmol of dimetbyldiosirane respectively then bested with piperidine; lanes 10 and 15: same as lanes 9 and 14 but without plperidine treatment.

91

9

Vol.

169,

No.

1, 1990

and 11 to 14) dioxirane At the

with

a single

On treatment material strong

the

the

(gel 3'-end preference

not

that

4S-mer

labelled for

(lanes chain.

labelled

Identically

molecule

1

are at

c f

with

RESEARCH

concentration of

C

the

4

G

shown sites

G

234567

COMMUNICATIONS

of

cleavage

dimethylwith

the

9 and 14)

there

was a bias

cleavage

sites

were

Experiments

were

32P at either

and piperidine,

behaved

cleavage

the

multiple

dimethyldioxirane shown)

BIOPHYSICAL

same extent

oligonucleotide

stranded with

the used

indicating

into

twice

to effect

concentrations

framnts

AND

approximately

was required

introduced

for

though

hi&hest

shorter

BIOCHEMICAL

to the in Fig.

also

S.-end

34-mer

(Fig.

3 and likewise

of guanine

bases.

towards

being

its

the

duplex.

S'-

conducted or 3'-end.

labelled 2).

Results

illustrate The even

8 9101? G A*,G CGT G AC TGG C G ET GT T G

I.

ii G G

,

C T G ,.,

C A C A G T A G G

Fix. 3. Autoradiograph Of single stranded /3'-32Pl end-labelled 45-mer treated with dimetbyldioxirame and piperidine. Lanes 1 and 2: uncut 45-r lanes 3 to 6 and 11: products of before and after beating with piperidiee; Waxsm-Cilbert sequencing reactions as indicated; leaes 7 to 10, 45-mer (20 DNA (4 ~6) reacted with 4, 7. 14 and 22 nmol of pool) plus carrier dimethyldioxirane respectively then heated with piperidine.

92

a

Vol.

169, No. 1, 1990

density

of

context

has

the

bands

to

those

are

modified

that,

in

it

is

to

some

on The

sulphate

(17).

offers

several entails

the

or to

solutions.

totally

reagent

(9)

has

which

a half-life Other

DNA

at

acetone

is

stable 48

methods

guanine

photolysis

that

addition

can

be

laser

found

for

have

chemical DNA

by

at

proteins

and

potential

chemical

uncharged

248

of

has small

Acknowledgments: Dr R.R. RcGuckin

molecule

no

necessity

the

sample

by

of

dimethyl

dimethyldioxirane fewer for

is

ethanol

is

is

because

and

oxygen,

noteworthy

prepared stored

waste

avoided

acetone It

without

supernatant

agent

volatile

reagents

achieved

radioactive

when

reported

to

treatment blue

nm

at

and

that

and

inexpensive

-7OOC;

at

the

To

sequence with

bases the

with

diethylpyrocarbonate

2S°C,

it

these

secondary

structure

in

DNA-protein

or

was

to

be

properties readily

and

93

this

of

the

by SERC preparation

not

used

as

a

recognition

of

towards respect.

As

local

complexes,

being in

the

but

reactivity in

DNA-ligand of

soluble

supported with the

DNA widely

its

useful

or intensity have

the

upon

DNA

is

of

investigating

Depending prove

high

approaches

is

of (la),

(20)

with

determination

for

cleavage

methylamine

date,

also

which

predominant

excitation

may

desirable

This work for assistance

to

dimethylsulphate,

reagent

of

lead

(19),

and

(22).

molecules.

probe guanine

months

(21).

cosxnon

other

dimethyldioxirane

use

is

a readily

and labelled

a

requires

heating.

methylene

‘footprinting’

proteins,

dimethyldioxirane

many

a simple

DNA

of

to

gentle

its

in in

or

chain

chemical

a quenching

is

been

of

application

probe

environment

of

dication radiation

routine

to

terminally provides

the the

of

decomposed

include

diethylpyrocarbonate,

methods,

generation

by

it

time-consuming,

solution

dimethyldiazaperipyrenium pulsed

such.

of

There

or

in

guanine--specific

less

the

presence

the

appear rise

h.

residues in

thymine,

would give

affords

bases

As

recovery

simply

in

of

than

deoxyribo-

it

not

clearly

guanine

these

is

salt

removed,

dimethyldioxirane

DNA

especially

so, do

modification

with

added

dimethyldioxirane be

above

steps.

Furthermore,

excess

If

sequence.

quantitative

of

DNA,

be

of

individual

in

By

should

fragments

the

lesions

conventional

It

that

it

longer of

of

utilise

operational

so

complications

thus

the

comparison

fewer

sequence

guanines.

concentration for

resultant

unknown

advantages.

precipitation

base

individual

nucleobases

positions

which

In

that

of

described

the

known

procedures

suggests

direthyldioxirane. the

RESEARCH COMMUNICATIONS

piperidine.

alternative

sequencing

gel

reactivity

other

by

mapping

of

practicable

the

protocol

for

fragments

of that

with

the reactivity

conditions

guanine,

experimental

method

view

degree

beating

on

dimethyldioxirane

probable

to

AND BIOPHYSICAL

the

cleavage

In

contrast

scission

the

the

here.

nucleosides,

lanes on

of

optimise

studied

and

the

influence

adjustment

feasible

DNA

in

little

appropriate

rapid

BIOCHEMICAL

a highly aqueous

grant of

reactive, mediaatneutralpH

GR/B 48060. dimethyldioxirane.

We

thank

a

Vol.

169,

No.

1, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERRNCES 1. 2. 3. 4.

5. 6. 7. a. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

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