ANALYTICAL
BIOCIiEMISTRY
RNA-RNA
467-476 (1972)
50,
Hybridization Containing
ROLAND Institut
fiir
Biologic
in Aqueous Formamide
FRIEDRICH III
Freiburg, Received April
(Genetik Freiburg
Solutions
AND
und
GtfNTER
FEIX
Molekularbiologie),
i. Br.,
Universitiit
Germany
12, 1972; accepted July 17, 1972
The study of nucleic acid interactions by assays as RNA-DNA hybridization and DNA renaturation has found widespread use in molecular biology (1). These reactions are mostly performed at elevated temperature, preferentially some 25°C below the midpoint of the thermal transition (T+,J, in order to achieve a maximum rate of reaction (2). If, however, these reactions are carried out in aqueous solutions of various organic solvents which lower the melting temperature of double-stranded polynucleotides, hybridization will take place at lower temperatures, thus reducing the danger of chain scission and depurination during prolonged incubations (3). Formamide was found to be particularly useful for this purpose (4) . This communication shows that formamide may also be used with advantage for RNA-RNA hybridization in solution at low temperature. Using the renaturation of denatured double-stranded phage RNA as an example, various parameters of this reaction have been measured to explore the properties of this system and to evaluate the optimum conditions for the annealing reaction. It will be shown that t.he integrity of the RNA is preserved during this treatment and that well-matched doublestranded RNA is formed during the annealing in the presence of formamide. MATERIALS
Salts were reagent grade form Merck (Germany), Formamide (purchased from Merck) was used without further purification since the infectivity of phage RNA incubated for 12 hr in 40% formamide was not affected (see “Methods”). Diethylpyrocarbonate was a gift of Bayer Leverkusen (Germany). Pancreatic RNAse was obtained from Biihringer (Germany). Viral RNA of phage MS2 or Q/3, labeled or unlabeled, was prepared as outlined by Weissmann and Feix (5). Double-stranded RNA of phage 467 Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Q,j3 or MS2, labeled or unlabeled, was prepared as described by Pollet et al. (6) omitting the elution step with 30% alcohol from the cellulose column. In large-scale preparations, the double-stranded RNA eluted from the cellulose column was concentrated by ultrafiltration in the Amicon apparatus using the PM-30 filter. The mixture of partially and entirely double-stranded RNA obtained by this procedure was used as such and in the following is referred to as double-stranded RNA. 32P-Labeled double-stranded RNA was isolated from phage-infected Escherichia coli cells grown in low phosphate medium (7) in the presence of 10 &&‘rnl [32P]-phosphate. The double-stranded RNA (specific activity 1 X 106 cpm/pg) was 75% RNAse resistant (see “Methods”). This value dropped to 1.4% after heat denaturation for 1 min at 100°C. METHODS
Acid-insoluble and RNAse-resistant radioactivity were determined as described by Billeter and Weissmann (8). Sucrose density gradient centrifugation was carried out by layering the sample (diluted to 10% formamide) on a 5 ml linear 5 to 23% sucrose gradient containing 20 mM Tris-HCl buffer, pH 7.4 (23°C)) 100 mM NaCl, and 3 mM trisodium EDTA and running the gradient in a SW-65 rotor (Beckman Instruments) for 120 min at 64,000 i-pm. The infectivity assay for viral RNA was carried out according to Strauss (9) (1 pg RNA yielded lOlo to lOI plaque-forming units). Radioactivity measurements were made in a Beckman liquid scintillation spectrophotometer using a toluene-based scintillator for counting the membrane filters (Sartorius, Germany) or a dioxane-based scintillator for counting fractions of sucrose gradients. RNA concentrations were determined spectrophotometrically assuming an E (l%, 260 nm) of 250 dl gm-’ cm-l for single-stranded RNA and of 210 dl gm-l cm-l for double-stranded RNA (10). RESULTS
Denuturation
AND
DISCUSSION
of Double-Stranded Phage RNA in Aqueous Solutions of Formamide
Double-stranded phage RNA can be denatured in aqueous solutions at rather high temperatures only. In the case of phage MS2, for instance, at a salt concentration of 1 X SSC (0.15 1M NaCI, 0.015 M sodium citrate -(pH 7.0) ) , the T,. is found to be 103°C (10). This T,, can be shifted considerably to lower temperatures by denaturing the double-stranded RNA in the presence of dimethyl sulfoxide (11). The melting temperature can also be decreased by the addition of formamide as demonstrated in Fig. 1. This figure represents thermal denatura-
RNA-RNA
50
60
469
HYRRIDIZATION
70 tcmpcralure
80 w
so
IC
FIG. 1. Melting curves of double-stranded viral RNA in presenceof formamide: (0) 29% formamide, 1 x SSC (MS2 RNA); (0) 40% formamide,1 X SSC t&P RNA) ; (0) 43% formamide, 1 x SSC (MS2 RNA); (A) 60% formamide, 1 X SSC (MS2 RNA) ; (0) 40% formamide, $6 x SSC (Q/3 RNA) ; (A) 90% formamide, Ys X SSC (MS2 RNA). Individual 30 $1 samples containing 1 mM EDTA, 0.33 pg ‘H-labeled double-stranded MS2 RNA (1250 cpm/&, or 0.51 pg V-labeled double-stranded Q/3 RNA (12,100 cpm/ag) and formamide and salt @SC) at the concentrations indicated in the figure were heated for 2 min at the temperatures given in the figure. The samples were then rapidly cooled down, diluted to 1 ml
1 X SSC, and subjectedto RNAse digestionas describedin “Methods.”
tion profiles of 3ZP-labeIed doubIe-stranded phage RNA at different concentrations of formamide and salt. Individual samples of radioactive double-stranded RNA were heated at the temperatures indicated in the figure followed by a RNAse treatment at room temperature. The radioactivity in RNA became increasingly RNAse sensitive with the rise in temperature and thereby progressively acid soluble as the denaturation went to completion. Thus, the T, values for different samples can be directly deduced from the measurement of acid-soluble radioactivity as shown in the figure. The intactness of the RNA strands is not impaired by these denaturation conditions, as will be shown in Fig. 4 and Table 1. Annealing of Single-Stranded Viral RNA (“Plus” Strands) with Complementary RNA (“Minus” Strands) in the Presence of Formamide In order to evaluate suitable conditions for RNA-RNA hybridization reactions in the presence of formamide, denatured radioactive double-
stranded
RNA
was submitted
to different
annealing
conditions.
The
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amount of double-stranded RNA formed as given by its RNAse resistance under standard conditions was taken as a measure of the effectiveness of the various annealing conditions. The RNAse-resistant radioactivity of the double-stranded RNA before denaturation was defined as lOO(r, resistance. Figure 2 shows that, at a concentration of 40% formamide (Fig. 2a), at a salt concentration of 1 X SSC (Fig. 2b), and at 37°C using 40% formamide and 1 X SSC (Fig. 2c), a maximum of double-stranded RNA formation is reached corresponding to a yield of more than 90%. The same percentage of renatured product was obtained by annealing the RNA under standard aqueous conditions at 85°C (10). The rate dependency of the annealing reaction on the RNA cocentration
0
20 formamide
40 concentration
60
80 !*/A
FIG. 2. Extent of rem&ration at various concentrations of formamide (a> and salt (b), and at different temperatures (c) : (a) 30 ~1 samples containing 1 mM EDTA, 1 X SSC, 0.3 gg denatured “P-labeled double-stranded Q/3 RNA (10,700 cpm/#g) and formamide at percentage indicated in the figure were incubated for 12 hr at 37°C; after incubation the RNAse-resistant radioactivity of the assay mixtures ww determined as described in “Methods.” (b) 30 cl1 samples containing 1 mM EDTA, 0.3 pg denatured ‘“P-labeled double-stranded MS2 RNA (67,000 cpm/&, 40% formamide, and salt in amounts indicated in the figure were incubated for 12 hr at 37°C; after incubation the samples were processed as indicated in part a of the figure. (c) 30 ~1 samples containing 1 mM EDTA, 1 X SSC, 40% double-stranded Qp RNA (10,700 formamide, and 0.3 ~lg denatured “P-labeled cpm/gg) were incubated for 12 hr at temperatures indicated in the figure and further processed as indicated in part a of the figure.
RNA-RNA
5
15
FIG.
HYRRIDIZAl’ION
25 temperature 2
(continued).
35 (‘Cl
L5
472
FXEDRICH
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is shown in Fig. 3. At a concentration of 260 pg/ml, the hybrid formation is completed already after 1 hr of incubation at 37”C, whereas at lower RNA concentrations it takes several hours to reach the plateau. It should be noted that the double-stranded RNA preparation used contains slightly more phage ‘rplus” strands than ‘(minus” strands since the doublestranded RNA has not been separated from the partially double-stranded RNA carrying “plus” strands tails (12). From the above experiment the following conditions for the low temperature hybridization of RNA are suggested: 40% formamide, 1 X SSC (pH 7.0), 37°C and period of incubation depending on the RNA concentration. The renatured product is best isolated by first removing the formamide by dialysis since an alcohol precipitation in the presence of formamide may lead to aggregations. Properties
of the Renatured
Product
A sedimentation analysis of the components of the renatured product shows that the integrity of the RNA is fully preserved during the anneal-
0
1
2 time
of
3 incubation
4
5
6
(hl
FIQ. 3. Renaturation kinetics of denatured double-stranded RNA: (0) 4 pg RNA/ml; (0) 43 pg RNA/ml; (0) 60 pg RNA/ml; (A) 260 s RNA/ml. 30 gl samples containing 1 mM EDTA, 1 X SSC, 40% formamide, and denatured doublestranded Q,G RNA in amounts indicated in the figure (all individual solutions contain the same amount of =P-labeled denatured double-stranded RNA) were incubated at 37°C for the times indicated in the figure and further processed as indicated in part a of Fig. 2.
RNA-RNA
ing process. The RNA formed “minus” strands under standard formamide (see above), followed run through a sucrose gradient.
olA, I
, 5
10
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HYBRIDIZATION
by incubating full annealing conditions by denaturation in The single-stranded
--. , .?TYY-Y~
15 fraction
20
25
length “plus” and in the presence of 407% formamide, is RNA generated by
I
0
loo.1
FIG. 4. Sedimentation analysis of renatured product. A mixture of full-length “P-labeled Qp rrplus” and “minus” strands was isolated by purifying denatured double-stranded ($3 RNA (3800 cpm/pg) on a sucrose gradient, pooling the fractions corresponding to the leading edge of the 30s peak, and concentrating the RNA by alcohol precipitation. (a) 0.15 g of this purified RNA preparation was mixed with 1 erg ‘H-labeled MS2 RNA (2000 cpm/pg) and run in a sucrose gradient. Fractions were removed from the bottom and the radioactivity was measured as outlined in “Methods.” (--) “P-Labeled Qp RNA. (---) *H-Labeled MS2 RNA. (b) 0.3 pg of the purified RNA preparation was incubated for 6 hr at 37°C in a 15 gl sample containing 40% formamide, 1 X SSC, and 0.1% SDS. After this incubation tie mixture was heated for 2 min at 9O”C, cooled down rapidly, diluted with water to 100 ,u& mixed with 1 pg *H-labeled MS2 RNA, and layered on a sucrose gradient. The experiment, was continued as described in part, a. (-) PP-L&&d Q/3 RNA. (-- -) ‘H-Labeled MS2 RNA.
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the denaturation exhibits the same sedimentation behavior as does full length viral RNA. The S2P-labeled Q/3 RNA strands before (part a of Fig. 4) and after annealing followed by denaturation (part b of Fig. 4) show very similar sedimentation patterns, indicating that no degradation of the RNA took place. In the gradients SH-labeled MS2 RNA was included as internal marker. MS2 RNA sediments slightly slower than QJ~ RNA (13). The hybridizing temperature of 37°C which has been chosen to reduce breakage of RNA at prolonged incubations is below the temperature assumed to be optimal for hybridization (about 25°C below the T, (2) ) . Therefore it was necessary to examine whether the renatured product contained unpaired or mispaired bases. This was done by treating the renatured product with RNAse under conditions that will discriminate between single- and double-stranded regions of RNA by hydrolyzing singlestranded regions only (14). After such a RNAse treatment, the RNAse was destroyed by incubating the solution for 3 hr at 37°C in the presence of 0.06 1M diethylpyrocarbonate (15). The double-stranded RNA was then denatured and the infectivity of the single-stranded RNA was determined and compared to the infectivity of a denatured double-stranded Infectivity
TABLE 1 Assay of Different Q@ RNA Preparations Plaque-forming
RNA
QB RNA (-4 Double-stranded &a RNA (B) Double-stranded Qa RNA CC)
I (not heated) 1 x 10’0 <2 x lee
II (denatured) 1 x 10’0 1 x 106 2.5 x lo6
unite/ml III (RNAse treated, than denatured) <20 1 x 106 2.5 X 106
Solutions of Qfl RNA (A), double-stranded Qfi RNA isolated from infected E. cdi cells (B), and double-stranded &e RNA prepared from full-length RNA strands as described in Pig. 4b (C) were tested for infectious units in the spheroplaat assay (column I). Aliquote of these solutions were tested for infectious units after denaturation in 40% formamide for 2 min at 90°C (~lumn II) as well ae after the same denaturation preceded by RNAse treatment (column III). Theinfectivity assay is not inhibited by a formamide concentration ae high ae 10% (16). The RNAse treatment was carried out in 2 X SSC at a RNAse concentration of 0.1 &ml for 30 min at 25°C. After this incubation, diethylpyrocarbonate was added ?o a final molarity of 0.66 and the solution was incubated for 3 hr at 37°C. After adding formamide to a 6nal concentration of 400/ the solution wae heated for 2 min at 90°C.
RNA-RNA
HYBRIDIZATION
475
RNA which was not treated by RNAse. Table 1 shows that the infectivity of the viral RNA which was part of the renatured product was fully preserved during the RNAse treatment, indicating that the bases in the double-stranded structure are completely hydrogen bonded. The table also shows that under the conditions of the RNAse digestion the infectivity of single-stranded RNA is completely inactivated and that the conditions chosen for denaturation of double-stranded RNA do not impair the infectivity of viral RNA. It is concluded that the use of formamide in RNA-RNA hybridizations is well suited for a complete annealing of complementary phage RNA without subjecting the RNA to high temperature. If this technique is applied with a more complex set of various complementary RNA molecules, it may first be necessary to test whether a specificity of the annealing reaction similar to that demonstrated above with phage RNA can be achieved with other species of RNA molecules. SUMMARY
In the presence of 40% formamide, perfect hybridization of denatured double-stranded viral RNA is achieved at low temperature. The integrity of the RNA is completely preserved during this hybridization procedure. ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft (SFB 46). We wish to thank Drs. G. Hobom and R. Roychoudhury for helpful criticisms the manuscript.
of
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3. Hsss~ovrrs, T. T., Arch. B&hem. Biophys. E. P., 1. Mol. Biol. 4, 467 (1962). 4. HELMKAMP, G. K., AND P. 0. P. Ts’o, J. Amer. Chem. Sot. 83, 138 (1961). BONNER, J., G. KUNQ, AND J. BEKHOR, Biochemistry 6, 3650 (1967). MCCONAUGHY, B. L., C. D. LAIRD, AND B. J. MCCARTHY, Biochemistry 8, 3289 (1967). GILLESPIE, S., AND D. GILLESPIE, B&hem. J. 125, 481 (1971). 5. WEISSMANN, C., AND G. F’EIX, Proc. Nut. Acad. Sci. U. S. 55, 1264 (1966). 6. POLLET, R., P. KNOLLE, AND C. WEISSMAN, Proc, Nat. Acad. Sci. U. S. 58, 766 (1967). 7. WEISSMANN, C., private communication. 8. BILLETER, M. A., AND C. WEISSMANN, in ‘LProcedures in Nucleic Acid Research” (G. L. Cantoni and D. Davies, eds.), p. 498. Harper & Row, New York, 1966. 9. STRAUSS, J. H., J. Mol. Biol. IO, 422 (1964). 10. BILLETER, M. A., C. WEISSMANN, AND R. C. WARNER, J. Mol. Biol. 17, 145 (1966). 11. STRAUSS, J. H., R. G. KELLY, AND R. L. SINSHEIMER, Biopolymere 6, 793 (1968). 12. FRANKTJN, R. M., Proc. Nut. Aeud. Sci. u, $, 55, 150: (1966).
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13. FEIX, G., H. SLOR, AND C. WEISBMANN, Proc. Nat. Acad. Sci. (1967). 14. BISHOP, J. M., AND G. KOCH, J. Biol. Chem. 242, 1736 (1967). 15. &ERQ, B., Biochim. Biophys. Acta 294, 430 (1970).
16.
STRAUSS,
J. H.,
AND
R. L.
SINSHEIMER,
J.
Viral. 4, 711 (1967).
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