A new method for sequence determination of large oligonucleotides

Vol. 17, No. 4, 1964

A NEW

METHOD

BlOCHEMlCAL

FOR

Robert

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

SEQUENCE

DETERMINATION

OF

LARGE

OLIGON-UCLEOTIDES

W. Holley,

James T. Madison

and Ada Zamir

U. S. Plant, Soil and Nutrition Laboratory, Soil and Water Conservation Research Division, Agricultural Research Service, U. S. Department of Agriculture, and Department of Biochemistry, Cornell University, Ithaca, N. Y.

Received

August

The most sequence

27,

1964

promising

approach

of an RNA involves,

to the determination

as an initial

of the RNA, and the determination oligonucleotides tion

that

of the

the diquate

are

structures

is

the determination

nucleotides.

The present to these

applied

successfully

nucleotide

communication

methods

are

of many larger

describes

a method

The method containing

inade-

oligowhich

is

has been

from

five

to eight

residues.

is

the 3'-phosphate,

partial

nuclease, (Razzell

first

since

degradation degrades

treated this

and the 3'-end identifying identity

and establishes

(Privat

with

1959)

products.

snake

products

of each stepwise formed

of the nucleotide the

sequence

group

de Garilhe,

from

a series

inhibits

separated

degradation

product

by alkaline

et al.,

1957). an exo-

the 3'-end

the oligonucleotide.

smaller

by chromatography, is determined

hydrolysis. positions

to

the action

of successively

are

389

phosphatase

venom phosphodiesterase,

in successive of

alkaline

stepwise

and gives

These

the nucleoside

with

phosphate

the oligonucleotide

and Rhorana,

degradation

the

the determinaparticularly

but

oligonucleotides.

of snake venom phosphodiesterase Then,

methods,

straightforward,

cleavage

of the various

oligonucleotides,

to oligonucleotides

The oligonucleotide remove

present

of the structures

larger

the enzymatic

structures

With

of the smaller

for

applicable

of the

formed.

and trinucleotides,

step,

of the nucleotide

This down the

by gives chain

Vol. 17, No. 4, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

EXPERlMENTAL Approximately

0.5 mg of oligonucleotide

0.1 M pH 8.3 Tris line

(chloride)

phosphatase

(Worthington",

according

to Garen

incubated

1 hr at 37".

water

with

aqueous remaining

the aqueous phenol

solution

can be raised case

this

solution

is

lower

of phenol

five

extractions

with

layer

approximately

(To determine incubation

cent

solution

is

the exact

left amount

or,

titrator.)

solution

is

immediately

to a 0.35

X 30-cm

(Tomlinson

and Tener,

1962).

ducts

are eluted

from

60 ml each of 7 M urea

L/Trade the of

reader

the

column

names and company and do not

the product

listed

infer

any endorsement

by the U. S. Department 390

is

sufficient 30 per-

for

the

and the

of DRAR-cellulose

formed

acetate

included

15

a small

the digestion,

column

of

to for

there

gradient

sodium

names are

0.1 ml

The degradation

a linear

and 0.6 M pH 7.5

To

according

and 3 ml of 7 M urea,

with

in

ml (2 mg per ml)

if

After

with

7 M urea

or it

buffer,

to approximately

is mixed

in

with

added

of enzyme needed,

can be carried

g of urea

of

low,

at room temperature

solution

packed

0.4

is

purified

may be required,

in an automatic

added

extractions

oligonucleotide

(Worthington,

the digestion

completion

volume

may be required.)

0.02

the

and the ether

The pH can be left

and approximately

and column

oligonucleotide,

from

of 0.1 ml of 0.1 M pH 7.5 Tris

chloride

and the

(previously

The final (The

is

0.6 ml of

removed

ether,

0.5 ml.

the pH to 6 to 7.

of alka-

solution

with

are

evaporated.

of the dephosphorylated

1964),

trial

is

and the

0.8 ml of phenol

traces

snake venom phosphodiesterase

minutes.

with

ml of

on DEAR-cellulose

diluted

snake venom phosphodiesterase

of 0.1 M magnesium

Keller,

then

in 0.10

mg per ml)

is added,

is

times

by the addition less

ml (0.4

The last

the aqueous

and ether

which

three

by at least

with

1960)

The solution

water).

layer

0.10

rechromatographed

and Levinthal,

and is extracted

saturated

buffer,

is dissolved

profrom

in 7 M urea,

the benefit

or preferential of Agriculture.

and

of

treatment

BIOCHEMICAL

Vol. 17, No. 4, 1964

fractions

of approximately

are

combined,

after

the

solutions

are

and Sober, separated 0.5

applied

are

"desalted"

on small

degradation hydroxide

directly

[isopropyl

alcohol,

60; water,

Hershey,

Dixon

alcohol-HCl

[isopropyl

41; water sides

and Chase,

1953)]

and nucleotides as well

1941)]

are

in

as from

spectra

in

ml of are

to two-

phase

direction,

the second

after

0.05

alcohol-water-ammonia

the vapor

first

from

with

The

and subjected

concentrated

identified

(Rushizky

and the hydrolysates

paper

anxnonia

170;

columns

isopropyl

in the

alcohol,

to 250 (Wyatt,

matograms

using 40;

absorption,

are hydrolyzed

Ribbon

fractions

by evaporation.

18 hours,

to S & S 589 Green chromatography,

by ultraviolet

is recovered

at 37" for

paper

Appropriate

DEAE-cellulose

products

dimensional

RESEARCH COMMUNICATIONS

collected.

located

and the material

stepwise

N potassium

1.2 ml are

the peaks

1962),

AND BIOPHYSICAL

(cf.

and isopropyl

hydrochloric direction.

their

The nucleo-

positions

elution

acid,

on the

of the

spots

chro-

from

the

paper. RESULTS Application

of

a hexanucleotide (Apgar,

the method

and an octanucleotide,

Halley

and Merrill,

since

known

that

complete

it

contained

was isolated

Gp was the

gave adenosine,

nucleotide.

Degradation

from a takadiastase

the hexanucleotide

present

method

nucleotide.

establishing with could

was used Partial

degradation

snake

venom phosphodiesterase,

followed

according

to

of

RNA

one Gp and two Up's, RNase digest,

It

was also snake

391

known

was that

Ap was the 5'-terminal indicating

as ApUp(Cp,Cp,Up)Gp. sequence

of the

by chromatography

The

remaining

of the hexanucleotide

the above procedure

it

venom phospho-

RNase gave ApUp,

the

nucleotides.

cellulose,

that

be represented

three

the alanine

Tl

with

pancreatic

to establish

the sequences

be described.

of the hexanucleotide

diesterase

that

will

from

one Ap, two Cp's,

3'-terminal

degradation

of

obtained

1962),

The hexanucleotide and,

to the determination

with on DEAE-

, gave the pattern

shown

in

Vol. 17, No. 4, 1964

Fig.

1.

four,

BIOCHEMICAL

The peaks

three

numbered

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5, 4, 3 and 2 contained

and two negative

charges,

material

respectively,

with

five,

and represented

the

82 0 z

4

0. 4-

, 0

1 Tube

Figure 1. diesterase

Chromatography of products digestion of ApUpUpCpCpG.

dephosphorylated

hexanucleotide,

products

of

charges,

respectively.

formed

the degradation,

alkaline

that

both

for

carrying

hydrolysis,

of these

stepwise

were

consistent

the hexanucleotide products

matographic

conditions

tide,

had been separation

tionation

these

used.

required of these

Peaks

the

all

products

the data

structures.

analyses

The sequence (The

smaller

of these

sequence

have

required

of

stepwise

in peak 2 under

the

would

are ApUpUpC

nucleotide

identifications

to establish

indicating

had 3’-terminal

ApUpUpCpCpGp.

If

3 and 4 each gave,

nucleoside,

with

lost

and

and two negative

products

ApUpU and ApU were

the chrosmaller

of the oligonucleofurther

frac-

of peak 2.)

The octanucleotide it

with

charges,

mononucleotides

peak 4, and complete

was therefore

degradation

products

three

primarily

consistent

venom phospho-

negative

as the only

degradation

structures

snake

five

four,

cytidine

peak 3 and ApUpUpCpC for

of the peaks

carrying

venom phosphodiesterase.

The only

cytidine.

of partial

Peak 2 contained

by the snake

after

No.

was known

that

one Gp

contained was

at

two Ap’s, the 5 ‘-end 392

five

Gp’s

and the Up

and one Up, and was

at

the 3’-end.

BIOCHEMICAL

Vol. 17, No. 4, 1964

The complete

sequence

degradation graphy nine tide.

with

was established

RESEARCH COMMUNICATIONS

using

the present

snake venom phosphodiesterase

followed

of the products negative

AND BIOPHYSICAL

gave the pattern

charges,

Peak 7, with

was a small seven

shown

amount

negative

in Fig.

method.

by chromato2.

Peak 9, with

of the original

charges,

Partial

octanucleo-

was the dephosphorylated

I A.260

2

mp

0 W lu d

C 1

Tube

Figure 2. diesterase

Chromatography of products of partial digestion of GpGpGpApGpApGpU.

octanucleotide,

and peaks

dation

carrying

products

Only

respectively. of each peak, guanosine,

No.

sent

the 3’-end

that

the

five,

six,

were,

for

, guanosine

was obtained peaks

venom phospho-

successive

and three

stepwise

negative

degra-

charges,

on alkaline

hydrolysis

6, 5, 4 and 3, respectively,

and adenosine.

of each stepwise

sequence

four

one nucleoside

and these

adenosine

6, 5, 4 and 3 were

snake

degradation

of the octanucleotide

The nucleosides product,

repre-

and establish

was GpGpGpApGpApGpUp.

SUMMARY Partial

degradation

phosphodiesterase identification may be used

gives of the

to establish

of large a series terminal

oligonucleotides of stepwise

nucleosides

the sequence 393

with degradation

obtained

from

of the oligonucleotide.

snake venom products, these

and

products

Vol. 17, No. 4, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENT This

work

was supported

and by the National

Institutes

in part

by the National

Science

Foundation

of Health. REFERENCES

Apgar,

J., Holley, R. W., and Merrill, S. H., J. Biol. Chem. 237, 796 (1962). C., Biochim. Biophys. Acta, 2, 470 (1960). Garen, A., and Levinthal, Hershey, A. D., Dixon, J., and Chase, M., J. Gen. Physiol. 2, 777 (1953). Biophys. Research Commun. 17, 412 (1964)(This Keller, E. B., Biochem. Privat de Garilhe, M., Cunningham, L., Laurila, U.-R., and Laskowski, M ., J. Biol. Chem. 224, 751 (1957). Razzell, W. E., and Khorana, H. G., J. Biol. Chem. 234, 2114 (1959), Rushizky, G. W., and Sober, H. A., Biochim. Biophys. Acta, 55, 217 (1962). Tomlinson, R. V., and Tener, G. M., J. Am. Chem. Sot. 84, 2644 (1962). Wyatt, G. R., Biochem. J., 48, 584 (1951).

394

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