A method for the detection of RNA-DNA complexes

Vol. 12, No. 2, 1963

BIOCHEMICAL

AND BIOPHYSICAL

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AMRTRDDFORTBE DETRCTIONOl? RNA-DNAWHPLRXRS Agnar P. Nygaard* - Department of Microbiology and B. D. Hall - Department of Chemistry University

of Illinois

= Urbana, Illinois

ReceivedMay30,15%3 Although several methods have been developed to study the interaction tween DNA and RNA @all and Spiegelman, 1961; Bautz and Ball, McCarthy, 1962), none of them are suitable

cellulose membranefilters

1962; Bolton and

for detailed kinetic

attempt to find a faster and more quantitative

studies.

these filters

In an

method we discovered that nitro-

have absorption characteristics

that can be used for

the separation of free RNA from RNAbound to DNA. In salt solutions concentration

be-

of moderate

absorb heat-denatured DNAas well as complexes be-

tween DNAand RNA, whereas free RNApasses through the filter.

Thus the reaction

of labeled RNAwith unlabeled DNAcan be followed by measuring, in samples taken at different

times, the amount of radioactivity MATERIALS

Nitrocellulose aqueous solutions, ell,

MembraneFiltere:

caught on the filter.

AND METHODS

--The filters

27 lll~~in diameter, and

were

used were Type A, coarse, for

obtained from Schleicher and Schu-

Keene, N.H. RN&--A culture

of TZ-infected

suspended in l/50 the original (PR 7.3)s and frozen. acidified fication on, the

After

E. &was

chilled

to O°C, centrifuged,

re-

volume of 0.01 M M~(OAC)~, 0.06 M RCl, 0.01 M Tria thawing, the suspension was stirred

3 min at 50°C,

with l/40 volume 0.1 N ROAc, and then subjected to the hot phenol puriprocedure (Scherrer and Darnell, RNA

1962).

After

the last phenol extracti-

solution was passed through a column of Sephadex G-25 (coarse, equil-

ibrated with 0.01 M KOAc-HDAcbuffer,

pH 5.2).

After

passage through the column,

*Visiting Professor at the Department of Microbiology Permanent Address: Nutrition Institute, University of Oslo, Blindem,

Norway.

Vol. t 2, No. 2, 1963

BIOCHEMICAL

AND BIOPHYSICAL

the RNAwas adjusted to pH 7.3, 0.5 M El, filters,

one filter

impurities DNA:

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and filtered

through nitrocellulose

per mg RNA. This step removes phosphorous-containing

which have a high affinity --DNA was purified

for nitrocellulose.

from phage T2 and T4 by the phenol extraction

procedure (Grossman, Levine, and Allison,

1961).

at 1OO'C for 15 min in 0.06 Ei KCl, 0.01 M Tris, ing from 0.6 to 150 pg/ml.

It was denatured by heating pH 7.3, at concentrations

vary-

The hot DNA solution was cooled by pouring directly

into l/3 volume ice, made from the ssme solvent,

at -3O'C.

A8SGRPTIONBEHAVIOROF NUCLEICACIDS ON NITBDCELLUlOSE =RANE m--Membrane

filters

do not appreciably

0.2 to .85 M KC1 (Fig. 1).

FILTERS absorb RNA in solutions containing

By washing with 60 ml of 0.5 HKCl,

7.3 absorption of RNA is further

O.OlM Tris,

pH

reduced to about 0.5%.

Heat-Denatured DNA:--The absorption of denatured T2 DNAby membranefilters is essentially quantity

complete over a wide range of salt concentration

of radioactive

DNA

absorbed is proportional

ed down to the smallest amount measured (Fig.

2).

(Fig.

The

to the amount of DNA filterThe proportionality

Above 1OOpg DNAper filter,

upward to about 200 pg DNAper filter.

1).

extends filtration

becomesvery slow.

Native DRAz--Native

T4 DNApreparations

absorption on nitrocellulose. preparation

absorbed varies

(10-50X), being least in the most extensively

The DNA fraction tion when filtered The relation

The fraction

are heterogeneous with respect to

which passes through the filter

a second time.

Upon heating,

of the transition

to

preparations. absorp-

this DNAis rendered absorbable. DNA

absorbed (Fig.

curve of DNA. In SSC buffer

is 85'C, as measured by either

(Murmur and Doty, 1959).

purified

shows no detectable

between temperature of heating and percent of

approximates the thermal denaturation

from preparation

filtration

3)

the midpoint

or hyperchromism

Vol. 12, No. 2, 1963

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ml SOLUTION

DNA.RNA

a5

0

COMPLEX 1.0

I05

:

M. KCI

Figure 1.

Figure 2.

Fig. 1.

X, andXNA,o-e, Absorption of denatured DNA, XTo each filter were on nitrocellulose membranefilters. 89 jzg heatadded 2.5 ml of a solution containing: denatured unlabeled T4 DNA; 9.Opg heat-denatured P32 T2 DNA, 40,000 q&g; 43 ug Ii3 T2 RNA, 680 cpn/cJg (labeled from O-5' after T2 infection); KC1 was added All samples were to give the indicated concentration. buffered with 0.01 M Tris, pH 7.3. The filters were not washed as in the regular procedure.

Fig.

Absorption of denatured DNAand of DNA-FWAcomplex on DNAwas nitrocellulose membranefilters in 0.5 M El. a mixture of T4 DNA and T2 DNA (~32, 35,000 cpm/pg, in the ratio 250/l. The DNA-RNAcomplex was formed from heat-denatured T2 DNA, (24 pg/ml) and 15-19' P32-T2 RNA, 14.1 pg/ml, 13,000 cp&g, incubated for 70 min. at 65-67'C in 0.5 M KCl, O.OlM Tris, pH 7.3.

2.

RNA-DNACOMPLgXFORMATION When incubated with denatured T2 DNAin saline solution, converted to a form which is absorbed by membranefilters

TZ-specific

(Fig. 4).

RNAis

Several

lines of evidence suggest that this change in absorption behavior is a consequence of RNA-DNAcomplex formation: 1.

The process requires elevated temperature (>40°C) and moderately high

ionic strength

(>O.l

M), as do the recombination of complementary DNA 1M

BIOCHEMICAL

Vol. 12, No. 2, 1963

loo

0

OF T4-DNA AS FUNCTION OF TEMPERATURE PRE-HEATING

I 80

85 TEMPERATURE,

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A OF

I

I I

so

95

lC

“0

loo MINUTE6

(at

200 65-C

Figure

Figure 3.

300 1

4.

Fig. 3.

Absorption of T4 DNAon nitrocellulose membranefilters after heating the DNAto various temperatures. T4 DNA, which had been passed through membranefilters in order to remove all absorbable DNA, was diluted into 0.1511NaCl, .O15 M Na citrate buffer to give a final concentration of 53@g/ml DNA. After heating for 15 minutes at the indicated temperature the solution was fast cooled and diluted to give 24 pg[ml DNA in 0.5 M KCl, O.OlM Tris, pH 7.3; 4 ml of this solution were added to the filter. The absorption to the filter was computed from the extinction of the filtrate at 260 x+~.

Fig. 4.

The Dependenceof RNA-DNAcomplex formation on denatured DNA. X, O-5' ?I3 T2 RNA, 5.2 ug/ml, 680 cpm/pg; xnative T4 DNA, 20 w/ml, 45%of which was absorbed on the filter in the native state. @-a, DNAheattreated fo 15 minutes at 100°, otherwise as X-X; Q-Q, H5 T2 RNA, 5.2 pg/ml, 680 cpm/ug.

strands (Marmur and Lane, 1960) and the formation of RNA-DNAhybrids and Spiegelman, 1961).

In 0.5 M ICCl, the rate

sured by the filtration

technique, is maximal at 67'C.

2.

Incubation of T2-specific

of complex formation,

as mea-

$NA under these conditions produces no filter-

absorbable RNAunless heated T2 DNAis present (Fig. 4). the native DNApreparation

(Hall

Although 50%of

tested was absorbed, RNAwas not complexed by it. 101

Vol. 12, No. 2, 1963

3.

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AND BIOPHYSICAL

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Over a lOO-fold range of the concentration

which RNAbecomesabsorbable is proportional

of each reactant,

the rate at

to the product (RNA concentra-

tion) x (DNA concentration). In order to determine whether the RN&DNAcomplexes absorbed by membrane filters

are of a specific

nature, heterologous RNA-DNAmixtures were tested. readily

Pulse-labeled RNA from T2 infected &

formed complexes with denatura

ed DNAfrom either T2 or T4, but did not react detectibly 2. cereus, g. subtilis,

or calf thymus.

sources is efficiently

the isolation cleotide

of T4-specific

u,

(Denatured DNA from each of these

absorbed by the filter).

is the fact that complex formation,

with DNA fromg.

Further evidence for specificity

filtering,

and elution

RNAof essentially

of the complex permits

100%purity,

as judged by its nu-

composition (Cl@--15X, A&P--32%, DMP--31%, C&@-21%).

DETRCTION OF RNA-DNAm

BY FILTRATION

Remwal of Free RNAr--The major problem in using absorption on nitrocellu---lose to detect RNA-DNAcomplexes has been to reduce the "background" caused by trapping of free RNAand/or other labeled substances.

This background has been

reduced to 0.5% (0.5% of the labeled RNA passing through a filter adopting the following

measures: soaking the filters

use (soaking in water is not effective), absorption,

and filtration

and Methods).

decrease in

in KC1 solution

prior

extensive washing of the filter

of the RNAprior

Somefurther

is caught) by

RNA

to

after

to complex formation (see Materials absorption can be obtained by washing

at higher temperature (6OOC). ReproducibilitY

of the Method: --When the foregoing precautions are observed, ---

there is both reproducibility between the quantity filter

(Fig.

2).

between replicate

proportionalit

of RNA-DNAcomplex in the sample and the radioactivity

When conditions

plexed is small (uniformly

are such that the fraction

labeled RNA, or high RNA/DNAratio),

complexes may be made difficult sorption.

samples and strict

by the variable

In such circumstances, proportionality 102

of th

of labeled RNA comdetection

of

background caused by free RNAabof the results obtained with

BIOCHEMICAL

Vol. 12, No. 2, 1963

AND BIOPHYSICAL

equivalent

samples of varying

legitimate

complex formation from RWA%oisen.

Absorption-Elution to the filter

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size provides a useful criterion

for distinguishing

Properties of Complexes.---Absorption

from 0.5 M KC1 solution

is quite insensitive

of RNA-DNAcomplex to pH in the range

9>PR>5. At pH 7 there is no elution

of the complex at room temperature even when 200

ml of the KC1 solution is passed through the filter; above 60°C.

If

the KC1 concentration

can be eluted without

0.01 M Tris,

pH 7.3.

dissociation

The elution

starts

to occur only

is lowered to 0.05 M, absorption of the com-

plex is incomplete even at room temperature. a filter

elution

RWA-DNAcomplex already absorbed on by passing through the filter

is more complete if

the filter

2 ml of

has been soaked

only a few seconds before use. Detailed Procedure --for Routine Analysis. *--The sample, which may vary from lop1

to 5 ml depending upon the radioactivity

0.5 M KCl, 0.01 M Tris,

pH 7.3, at room temperature.

this temperature no further

reaction

be done at short intervals

approximately

proximately stainless The filter dried,

In the diluted

(20 seconds or less).

10 ml of the samesolution

for at least 10 min before use, and

is passed through the filter

Suction is provided by an aspirator,

15 mmRg.

The filter

steel cylinder is finally

placed in a vial

state at

whenever time permits, and sampling can

is soaked in the KC1 solution

sample is applied.

into 15 ml of

occurs and the hybrid is stable for at least

24 hours; thus the sample can be filtered

The filter

of RNA, is diluted

before the

giving a vacuum of ap-

is mounted on a stainless

steel grid with a

placed on top as described by Roberts et al.

(1957).

washed with 60 ml of the KC1 solution at room temperature, with scintillating

solution,

and counted.

ACKNOWLRDGMENTS

Wewish to thank Mrs. Kathleen Kliewer for her assistance in these experiments.

This work was supported by grants from the Office

the U.S. Public Health Service (Grant No. A 3086).

One of

of Naval Research and US

(A.P.N.)

would like

Vol. 12, No. 2, 1963

BIOCHEMICAL

to express his gratitude University

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

to Dr. .H. 0. Ralvorson for arranging

his visit

to the

of Illinois.

Bautz, E.K.F.,

and Hall,

B.D., Proc. Nat'1 Acad. Sci. U.S., 48, 400 (1962).

Bolton, E.T., and McCarthy, B.J., Proc. Nat'%. Acad. Sci. U.S., 48, 1390 (1962). Grossman, L., Levine, S.S., and Allison, W.S., J. Mol. Biol., 2, 306 (1960). Hall, B.D., and Spiegelman, S., Proc. Nat'l. Acad. Sci. U.S., 47, 137 (196%) Marmur, J., and Doty, P., Nature, 183, 1427 (1959). Roberts, B.B., Abelson, P.H., Cowie, D.B., Bolton, E.B., and Britten, R.J., "Studies of Biosynthesis of Escherichia &," Carnegie Institution of Washington, Publication No. 607 (1951), p. 12b. Scherrer, K., and Darnell, J.E., Biochem. Biophys. Res. Coma., 7, 486 (1962). Marmur, J., and Lane, D., Proc. Nat'l. Acad. Sci. U.S., -46 453 (1960).

1OA