The binding of RNA to various celluloses

Vol. 50, No. 2, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRE BINDING OF RNA TO VARIOUS CELLULOSES Joseph DeLarco and Gordon Guroff Section on Intermediary Metabolism Laboratory of Biomedical Sciences National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20014 Received

December

4,1972 SUMMARY

Several celluloses were tested for their ability to bind homonucleotide oligomers and natural RNAs. Columns of these celluloses were run under conditions favoring hydrogen bond formation (neutral pH and high salt concentration). The materials binding at the high salt were released by eluting the columns with solutions of lower ionic strength. The binding to unesterified cellulose was much less specific than that found with celluloses esterified with oligo(dT) or oligo(dC) residues. The different cellulose preparations were quite different in their ability to bind the oligonucleotides and the RNAs. These variations suggested that an impurity present in the cellulose in varying amounts rather than the cellulose itself, is responsible for the binding properties of these samples. Treatment of the celluloses with sodium bisulfite reduced the amount of poly(A) binding which suggests that the binding is due to a ligninlike contaminant. Within using

the

last

few years

oligo(dT)-cellulose

RNA from

eukaryotic

a poly(A)-rich oligo(dT)

cells

for cells.

region

using

shown that

to the reports

difficult

to understand.

property

reports

of cellulose,

ability

to bind

natural

RNA.

variations

the

in

(6-9). and is

have

binds

to determine

as well

as poly(I),

indicate to bind

Copyright 0 1973 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

on the

that

different

homopolymers

poly(A)

466

not

recently

to oligo(dC)-

have

seemed

is

tested

and different

this

the

eukaryotic

we have

binding

celluloses

of

with

cellulose

were

and that

presence

column.

Since

to untreated

poly(U),

on the

but

celluloses

and messenger

RNA from

(10-12).

if

(l-5)

"hybridizes"

of messenger

binding

of different

ability

retained

poly(A)

to oligo(dT)-cellulose,

a number

The results

This

published

nuclear

RNAs depends

appeared

of messenger In order

poly(A)

their

of these

molecules

cellulose

of methods

of heterogeneous

of the purification

"messenger"

(13),

been a number

isolation

cellulose

cellulose

have

The isolation

unesterified brain

the

in these

esterified

More recently,

there

a general for

their

types

of

large

binding

is

not

speci-

BIOCHEMICAL

Vol. 50, No. 2, 1973

fit

for

data

poly(A),

suggest

but

that

also

extends

the binding

AND BIOPHYSICAL

to at least

is

RESEARCH COMMUNICATIONS

one other

polypurine,

due to a lignin-like

impurity

poly(1).

The

in some of the

celluloses. MATERIALS CF-11 and CC-41 celluloses lulose

was purchased

obtained

from

was obtained

the various

into

in minimum The column

were

with

washed

of deionized

water.

for

starting

volumes was washed

using

Aquasol

ribosomal

use by washing

was freed

cellulose.

E. ca

The cellulose the appropriate

in Table were

3

products

Nuclear)

enzyme with

low

and, salt

The samples containing

Then the columns finally,

with

buffer

3 ml

and the water

The columns

by determining

RNA or the 4000

series

were with

rethe

ribosomes

RNA was a gift Pronase,

100 mg of cellulose

487

radioactive

obtained

it

a

homopolymers

scintillation

described

by Jarlstedt

by passing

the A260 with

spectrometer

solute.

as previously

as described

with

same buffer.

by the column.

as a counting

from brain

was treated

M, pH 7.4)

pH 7.4,

measured

in a Packard

ribosomal

wool.

of

buffer.

were

of messenger

100 mg portions

2 ml of 0.1 M KOH and reequilibrated

RNA was prepared

preparation

were

CF-11 Whatman cellulose

glass

(0.01

by the

bound

The radioactive

RNA was prepared

with

buffer

buffer,

starting

by counting

brain

plugged

off

from

made by pouring

3 ml of the

washed

with

The RNA was extracted

(15).

with

to be the amount

(New England

The bulk

pipettes

M Tris-HCl

The material

spectrophotometer. quantitated

shown

homopolymers

prepared

were

of Tris-HCl

The RN4 and the homopolymers

were

were

Pasteur

0.5 M salt-containing

Gilford

38 cel-

and poly(1)

the experiment

The radioactive

The columns

3 ml of 0.01

was considered

generated

(14).

celluloses

0.5 M KCl.

for

poly(U),

type

Inc.

of Gilham

applied

Poly(A),

used

Laboratories.

Sigmacell

from Whatman.

Co.

and oligo(dC)-celluloses

by the method

washes

The poly(A)

Bio-Research,

Oligo(dT)-

obtained

Sigma Chemical

Calbiochem. from Miles

of Schwartz

were

from

were

AND METHODS

over

from Dr. RNase,

(13).

The brain

by the method and Hamberger a large Alan

column

of Murthy (16).

The

of oligo(dT)-

Peterkofsky.

or DNase by incubating

in a 1 ml volume

for

1 mg of

60 min at room

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

Vol. 50, No. 2,1973

temperature. portions

Treatment

of the cellulose

of the cellulose

cellulose

while

still

with

NaHS03 involved

in 5 ml of 1 M NaHS03 overnight

hot.

The exact

was measured by the dry weight

500 mg

and then filtering

amount of cellulose

of an appropriate

autoclaving

the

in each of the columns

portion

of the material

used to

pour the coluw. RESULTS The various homopolymers expected

preparations

(Table

1).

homopolymer;

bound poly(1).

oligo(dT)-cellulose

the condensing

that

the treatment

poly(u).

procedure

itself

capacities

celluloses

bound poly(A),

column retained

about the same amount of the polymers showing

binding

AS can be seen, the substituted

Neither

been run through

examined have different

for the

bound the

and oligo(dC)-cellulose

The W-11

cellulose

which had

in the absence of any nucleotide

that

the untreated

CF-11 cellulose

did not change the binding

bound bound,

properties

of the

cellulose. The several binding

properties.

poly(A)

capacity

Sigmacell

CF-11, CC-41,

of CF-11;

celluloses

The binding

for that

purpose.

to the columns the results cellulose

hold all

for

however,

poly(A)

all

the appropriate

because the

with

None of the

to the oligo(dT)-cel-

than the CF-11 specifically

When small amounts of labelled were consistent

different

had about 5x the

as did the (X-11.

bound more poly(1)

column bound essentially

columns will

but Sigmacell

as much poly(1)

cellulose

38, had widely

was not specific,

were equal in capacity

but the Sigmacell

esterified

and Sigmacell

None of them bound poly@)

also bound about twice

unesterified lulose

celluloses,

polymers

were added

the data in Table 1.

the radioactive

material

The oligo(dT)-

showing

polymer passed over them until

that

these

the capacity

of the column is exceeded. When the bulk RNA from the brain was passed over the columns, was retained which binds

(Table 2).

to oligo(dT)-cellulose

to oligo(dC)-cellulose Sigmacell

Labelled

brain

RNA has been

(13).

nor to unsubstituted

38 bound significant

quantities

Clearly, (Z-11

shown

this

and the binding

488

to contain

material

cellulose.

some material material

does not bind

Rut the CC-41 and was in rough propor-

to each to each to each

iAdded Added 3Added

A260 units A260 units A260 units

1.83

1,98

Untreated cellulose (Sigmacell-38)

1.33

1.26

1.05

1.06

Untreated cellulose (CC-41)

0.48

1.32

0.90

A260 units bound

0.54

cellulose (CF-11)

Different

Poly(I)*

to the

Untreated cellulose (CF-11)

Treated

0.60

Oligo(dC)-cellulose ((x-11)

4.52 2.40 4.12

of Homopolymers

Poly(A)' A260 units bound

Binding

3.56

1.

Oligo(dT)-cellulose (m-11)

Column

Table

column column column

Celluloses

0.28

0.37

0.22

0.21

0.36

0.33

Poly(u)3 units A260 bound

1,890

Untreated cellulose (Sigmacell-38)

Cellulose8

0,20

0.34

0.37

0.11

0.23

0,38

ribosomal RNA2 A260 units bound

Brain

of RNA to the Different

0.19

0.28

0.30

0.31

0.32

0.34

ribyomal RNA A260 units bound

E. &

'Added 9850 cpm, 3.74 A260 units to each column 2Added 7.63 A260 units to each column 3Added 7,65 A260 units to each column

1,107

Untreated cellulose (CC-41)

519

492

3,411

561

cellulose (CF-11)

Binding

Labellei brain RNA cpm bound

2.

Untreated cellulose (CF-11)

Treated

Oligo(dC)-cellulose @F-11)

Oligo(dT)-cellulose @F-11)

Column

Table

BIOCHEMICAL

Vol. 50, No. 2, 1973

Table

Effect

3.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of NaHS03 Treatment on the Binding the Different Celluloses

to

Autoclaved' with 1 M NaCl

Autoclaved' with H20

Column

of Poly(A)

POSY (A)

2

A260 units

Autoclaved' with 1 M NaHS03

bound

Whatman CF-11

0.46

0.40

0.21

Whatman CC-41

2.57

2,58

0.64

Sigmacell-

3.23

3.24

0.92

1

500 mg of the cellulose was autoclaved (20 lbs, 121') for 16 hours in 5 ml of the appropriate solution, filtered while still hot, and washed with hot water and with ethanol.

2 Added

tion

to the binding

ribosomal

for

ed the

capacity

materials.

to any of the preparations

poly(A),

to bind

to each column

by these

of the Sigmacell

capacity

ability

A260 units

of poly(A)

RNA bound

Treatment ing

3.60

either

On the

of this

to bind

cellulose

the material

Pronase,

the homopolymer.

brain

nor E* coli

used.

38 cellulose,

with

Neither

RNase,

other

hand,

poly(A)

with

the highest

or DNase did treatment

by more than

not

with

bind-

lower

its

NaHS03 lower-

70% (Table

3).

DISCUSSION The data cellulose data

presented

will

bind

have been

(personal poly(A), lulose

but

does not

is

not

at all

of value

substantially

the

reports

and messenger

exactly

and directly

The binding

in the

to these

celluloses.

of choice

by Drs.

since

appropriate

purification

because

that

untreated

RNA from

appears

seem to be specific, The use of the

seem to be the method is

confirms (11)

comnication).

probably

ity

poly(A)

confirmed

preparations.

bind

here

Haim Aviv

of its

greater.

491

38

(12).

These

and Philip

to be due to the presence poly(1)

binds

untreated

of messenger However,

eukaryotes

Sigmacell

specificity

cellulose

RNA since

oligo(dT)-cellulose

to the

Leder of the

same celpreparations

ribosomal

RNA does

binding and because

its

would capac-

BIOCHEMICAL

Vol. 50, No. 2,1973

The binding of poly(A) be due to the intrinsic

AND BlOPHYSlCAt

sequences to untreated celluloses does not seemto

properties

of the cellulose

treatment with NaHS03lowers the capacity ration

classic ways to remove lignin

itself.

suggests that an impurity

lignin,

or of lignin-like

mines the ability

in the prepa-

from wood pulp (17), the data indicate

itself

or to somelignin-like

material.

polymer of aromatic residues, and, thus, is an appropriate binding of polynucleotides,

The observation that

Since treatment with NaIlSO is one of the

is responsible for the binding.

binding is due to lignin

RESEARCH COMMUNICATIONS

more specifically, materials,

polypurines.

that the

Lignin is a

candidate for the The amount of

contaminating the various celluloses deter-

of these preparations

to bind messengerRNA. The capacity

of

these materials to bind polypurines rmst be enormous since the contaminations found in these highly purified

samples should not be above trace levels. ACKNOWLEDGEMENTS

The help and advice of Drs. Haim Aviv and Philip

Leder is such appreciated.

Conversations with Dr. Todd Miles are responsible for the suggestion that lignin might be involved

in the binding. REFERENCES

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17.

Nakazato, H. and Edmonds, M., J. Biol. Chem., 247, 3365 (1972) Edmonds, M., Vaughan, M. H., and Nakazato, H., Proc. Nat. Acad. Sci. USA68, 1336 (1971) Swan, D., Aviv, H., and Leder, P., Proc. Nat. Acad. Sci. USA 69, 1967 (1972) Aviv, H. and Leder, P., Proc. Nat. Acad. Sci. USA69, 1408 (1972) Armstrong, J. A., Edmonds, M., Nakazato, H., Phillips, B. A., and Vaughan, M. H., Science 176, 526 (1972) Molly, G. R., Sporn, M. B., Kelly, D. E., and Perry, R. P., Biochemistry 11, 3256 (1972) Darnell, J. E., Phillipson, L., Wall, R., and Adesnik, M., Science 174, 507 (1971) Mendecki, J., Lee, S. Y., and Brawerman, G., Biochemistry ll, 792 (1972) Lee, S. Y., Mendecki, J., and Brawerman, G., Proc. Nat. Acad. Sci. USA68, 1331 (1971) Sullivan, N. L. and Roberts, W. K., Fed. Proc. 30, 1302 (1971) Kitos, P. A., Saxon, G., and Amos, H., Biochem. Biophys. Res. COIIIII.47, 1426 (1972) Schutz, G., Beato, M., and Feigelson, P., Biochem. Biophys. Res. Conm.49, 680 (1972) DeLarco, J. and Guroff, G., Biochem. Biophys. Res. Comn. In Press Gilham, P. T., J. Am. Chem. Sot. 86, 4982 (1964) Murthy, M. R. V., J. Biol..Chem. 247, 1936 (1972) Jarlstedt, J. and Hamberger, H., J. Neurochem. l& 921 (1971) Brauns, F. E., "The Chemistry of Lignin" in WoodChemistry, L. E. Wise and E. C. Jahn (Ed.) Chapter 11, Reinhold Publishing Corp., NewYork, 1952, Vol. 1, 2nd Edition

492