Proc. Nat. Acad. Sci. USAVol. 72, No. 1, pp. 83-87, January 1975

A Routine Method for Protein-Free Spreading of Double- and Single-StrandedNucleic Acid Molecules

(electron microscopy/benzyldimethylalkylammonium chloride/single-double strand discrimination/protein-nucleic acid complexes)

H. J. VOLLENWEIDER, J. M. SOGO, AND TH. KOLLER

Institute for Cell Biology, ETH-Z-Honggerberg, 8049 ZUrich, Switzerland

Communicated by A. Frey-Wyssling, September 30, 1974

ABSTRACT A protein-free nucleic acid preparationmethod for electron microscopy is described. The basicprocedure is very similar to the classical protein monolayerspreading techniques. The carrier protein (usually cyto-chrome c) is replaced by benzyldimethylalkylammoniumchloride. Both the hypophase method and the micro-diffusion or droplet method can be applied with this com-pound. Unlike cytochrome c, benzyldimethylalkylam-monium chloride does not lead to any apparent thicken-ing of the nucleic acid strands. Partially denatured DNAspread with this reagent shows a loosened structure with afoamy appearance in the regions previously considered tobe "unmelted," which open up locally into melted loopsof different size. Specifically bound proteins, such asRNA polymerase on bacteriophage T7 DNA, can be de-tected unambiguously.

Nucleic acid molecules are elegantly prepared for electronmicroscopy by the spreading techniques (1) first described byKleinschmidt and Zahn (2), in which DNA has basic proteinattached to it and is adsorbed to a denatured protein mono-layer at an air-water interface. Since the protein (usuallycytochrome c) bound to the DNA obscures details, a numberof attempts have been made to obtain the same quality ofnucleic acid preparation, but without the use of carrier protein(ref. 3, for reviews see refs. 4 and 5). Protein-free preparationtechniques have been applied in efforts to develop methods forsequencing nucleic acids by electron microscopy (6) and instudies of the interactions of nucleic acids with proteins andother cellular components (5, 7-9). Recently, protein-freepreparation techniques have been used in attempts to quanti-tatively map sites of specific protein binding along nucleicacid molecules, as in the case of the interaction of Escherichiacoli RNA polymerase with DNA from bacteriophages T7(10-12) and T3 (11). In the latter applications the quality and/or reproducibility of the preparations were generally notsatisfactory for obtaining mapping data with an accuracy of afew nucleotides. In order to obtain such accuracy, large scalestatistical evaluation of very precisely collected data isrequired (13). This means that the nucleic acid preparationtechnique used must be very reproducible and that the yieldof analyzable molecules or complexes must be high. We haveshown (13) that these requirements can be met for double-stranded DNA by the intercalating dye method (11). How-ever, to our knowledge, no method for the satisfactory prepa-ration of single-stranded nucleic acid molecules has been de-scribed.

In this report we show that benzyldimethylalkylammoniumchloride (BAC) (14) can be used for routine protein-freespreading of single- and double-stranded nucleic acid mole-cules, yielding unfolded and unaggregated molecules with anarrow length distribution suitable for statistical evaluation.In a forthcoming paper (in preparation) we describe the bind-ing sites of Qj3 replicase on single-stranded Q,3 RNA.

MATERIALS AND METHODS

Benzyldimethylalkylammonium chloride (BAC), a mixture inwhich the n-alkyl group was C12H25 (60%) or C14H25 (40%) waskindly provided in solid form (91.2%) by Bayer, Leverkusen,Germany. Two hundred milligrams of BAC were dissolved in100 ml of formamide by vigorous stirring and kept at roomtemperature until used. For each experiment this solution wasdiluted 50-fold in the solvent to be used. All other reagentswere analytical grade and were obtained from Merck. Toexclude contamination, all solvents were passed throughMillipore filters, pore diameter 25 nm (250 A), prior to theaddition of nucleic acids or BAC. Phenol-extracted DNA ofE. coli bacteriophage T7 and E. coli RNA polymerase were akind gift of Dr. R. Portmann, Max Planck-Institut firBiochemie, Martinsried bei Munchen, Germany. This materialwas the same as that used earlier (13). Phenol-extractedRNA from E. coli bacteriophage Qj3 was given to us by Dr.C. Weissmann, Institut fur Molekularbiologie I, University ofZurich, Switzerland.For partial denaturation, T7 DNA in 0.01 M sodium phos-

phate buffer at pH 7.8 and 3.5% formaldehyde was heated for10 min at 630 and then quenched in ice. Two methods wereused for the denaturation of DNA: (1) T7 DNA was dilutedin a solution containing 25 MuM Tris HCl (pH 7.9), 4 Murea, and 99% formamide. Denaturation as measured by theincrease in absorbance at 260 nm was complete within a fewseconds. (2) T7 DNA was diluted in a solution containing 0.01M triethanolamine -HCl (pH 7.9) and 3.5% formaldehyde.This solution was heated for 10 min at 80° and was thenquenched in ice. The complex between RNA polymerase andT7 DNA was allowed to form and then separated from un-bound enzyme exactly as described previously (13). For theproduction of support films, carbon was evaporated from anelectron gun (15) onto freshly cleaved mica in a Balzers BAE300 evaporator equipped with a turbomolecular pump. Thefilm was floated off on water and picked up on 400-mesh grids.For the nucleic acid specimen preparation two methods wereused: (1) To 0.1 ml of a solution containing between 0.1 and

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Abbreviation: BAC, benzyldimethylalkylammonium chloride.

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84 Biochemistry: Vollenweider et al.

FIG. 1. The width of a single micrograph represents 140 nm (1400 A): magnification X240,000.(A) Double-stranded T7 DNA prepared by the droplet method from a solution containing 0.1 ug of DNA per ml, 2.5 X 10-3% BAC,

5.3 M urea, 1.3% formamide, and 3 mM triethanolamine, pH 7.9. No uranyl acetate staining prior to shadowing. Strand width: about5-6 nm (50-60 A).

(B) Same as in (A), but grids stained with uranyl acetate prior to shadowing. Strand width: about 7 nm (70 A).(C) Double-stranded T7 DNA spreading by the protein monolayer technique. Two microliters of cytochrome c (5 mg/ml) in a solution

of 4 M Tris-HCl (pH 8.2) and 0.1 AI EDTA were diluted in 100 Mul of T7 DNA (1 Mg/iml) in 0.01 M triethanolamine (pH 7.9). Hypo-phase: 0.3 M ammonium acetate (pH 7). Strand width: about 16 nm (160 A).

(D) and (E) Partially denatured T7 DNA, details of molecule represented in Fig. 3B. Strand width: singlestranded DNA about 4 nm(40 i&); double-stranded DNA about 10 nmn (100eA).

(F) Denatured, single-stranded T7 DNA. No uranyl acetate staining. Denaturation of the DNA at 800 for 10 min in a solution con-taining 0.01 M triethanolamine (pH 7.9), 3.5% formaldehyde and subsequent quenching in ice. Spreading was with the droplet methodfrom a solution as in (A). Strand width: about 2-3 nm (20-30 A).

(G) Same experiment as in (F), but with the preparations stained with uranyl acetate prior to shadowing.(H) Denatured, single-stranded T7 DNA prepared by the hypophase method. Spreading solution: 1 ug/ml of DNA, 2.5 X 10-3%

BACG 4 M urea, 8 ,M Tris HCl at pH 7.9, formamide 99%. Hypophase: redistilled water. No staining of the grids with uranyl acetateprior to shadowing. Strand width: about 6 nm (60 A).

(I) Same experiment as in (H), but with uranyl acetate staining prior to shadowing. Strand width: about 7 nm (70 A).(K) Denatured, single-stranded T7 DNA spread by the protein monolayer technique. Two microliters of cytochrome c (5 mg/ml) in

a solution of 4 M Tris HCl (pH 8.2) and 0.1 M EDTA were diluted in a solution of 100 Mul of single-stranded T7 DNA (1 Mg/ml) in 4 Murea and 99% formamide. Hypophase: redistilled water. Strand width: about 8 nm (80 A).

0.2 Mg of DNA or RNA in buffer, 0.2 ml of freshly diluted carrier protein was used. Spreading of the monomolecular filmBAC solution was added. The final BAC concentration was was visualized by sprinkling graphite powder onto the hypo-thus 2.5 X 10-3%. About 20 Mul of this nucleic acid-BAC phase. The DNA film was picked up on carbon-coated grids,mixture was spread onto a hypophase of redistilled water or washed on water for a few minutes, dehydrated in ethanol, andof ammonium acetate at concentrations up to 0.3 M. The air dried. (2) To 0.1 ml of a solution containing 0.01 ,ug ofspreading procedure was as described in ref. 1 except that no DNA or RNA in various aqueous solvents, 0.2 ml of freshly

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diluted BAC solution was added. Droplets of about 0.1 ml ofthis solution were placed on a fresh sheet of Parafilm or Teflon.The surface was protected from contamination by coveringwith a petri dish. After 10 min a carbon-coated grid wastouched to each droplet surface. The grid was then washed onwater for a few minutes, dehydrated in ethanol, and air dried.This procedure was similar to that in ref. 16, except that BACwas used instead of cytochrome c. After the DNA was ad-sorbed onto the carbon film, the specimens were rotaryshadowed at an angle of 70 with 1000 Hz carbon-platinum asmeasured on a quartz thin crystal monitor from Balzers. Inorder to thicken the DNA strands and thus enhance contrast,the specimens were occasionally stained with uranyl acetateprior to the platinum shadowing by immersing the grid for 15sec into a fresh solution containing 0.5mM uranyl acetate, 0.5mM HCl, and 90% ethanol. For the removal of excess stain,the grid was dipped into 90% ethanol and dried on filter paper.Electron micrographs were taken at 10,OOOX magnificationon a Siemens Elmiskop 101. The accelerating voltage was 100kV, the objective lens aperture was 36 Mm, and the condenserlens aperture was 200 Mm. For DNA length measurementelectron micrographs were enlarged lOX and were traced witha map ruler. The reproducibility of measurement was +-1%for double-stranded DNA and i3% for single-stranded DNA.

RESULTS AND DISCUSSION

In our first experiments, the BAC stock solution was preparedin water. The results obtained were unsatisfactory, however,in that the amount of DNA or RNA bound to the grid and theappearance of the nucleic acid filaments varied from one stocksolution to the next, possibly because of micelle formation.When we prepared the stock solution in formamide, however,the results became very reproducible. Double-stranded andsingle-stranded nucleic acids appeared as evenly distributed,unfolded, and unaggregated filaments on carbon as well as onParlodion-coated grids. No strands could be adsorbed to thegrids coated with aluminum-beryllium (17) or aluminumfilms (18).For double-stranded DNA specimens of equal quality were

obtained with the hypophase method and with the dropletmethod. There was a clear dependence of the appearance ofthe double-stranded DNA upon the salt concentration of thehypophase. When the hypophase consisted of redistilled water,the individual molecules were almost straight without bends,so that it was difficult to photograph an entire strand on oneelectron microscope plate. With increasing ionic strength ofthe hypophase, the molecules became more and more convo-luted. Hypophases containing 0.3 M ammonium acetateyielded well unfolded though frequently bent strands, whichstill could easily be measured with a map ruler.For single-stranded T7 DNA prepared with the BAC

droplet method the same number of broken strands wasobtained as with classical cytochrome c spreading experi-ments. However, with the BAC hypophase method morestrands were broken, indicating that the vigorous spreadingonto a hypophase may lead to disruption of the delicate singlestrands, which are probably less protected against breakagethan with cytochrome c. With single-stranded QB RNA,however, the difference between the droplet and the hypo-phase procedure was hardly noticeable. This more favorableresult is probably due to the smaller size (about 1.7 Mum) of thisRNA compared to T7 DNA (about 13 um).

Protein-Free Spreading of Nucleic Acids 85

100 A B C

50

10 15 10 15 10 15 gm

FIG. 2. Histograms. Ordinate: percent of total number ofmeasured molecules. Abscissa: Contour length of DNA moleculesin ,m.

(A) Double-stranded T7 DNA spread onto a hypophase of0.3 M ammonium acetate at pH 9 from a solution containing 1,ug/ml of DNA, 2.5 X 10-3% BAC, 13 mM ammonium acetate,7 mM Tris-HCl, pH 8.3, 77 mM KCl. Mean contour length:11.81 ± 0.3 ,m. Accuracy of measurement: ±4 1%.

(B) Double-stranded T7 DNA, conditions as in Fig. 1B.Mean contour length: 12.54 4 0.24 Mm. Accuracy of measure-ment: -1%.

(C) Single-stranded, denatured T7 DNA, conditions as inFig. 11. Mean contour length of apparently unbroken molecules:13.7 4± 1.1 rm. Accuracy of measurement: ±3%.

Fig. 1 shows the appearance of double- (A to C) and single-(F to K) stranded DNA under various conditions. For doublestrands (Fig. 1A and B) staining with uranyl acetate prior toplatinum shadowing had no significant effect upon thicknessand visibility. The width of these strands under our conditionsof platinum shadowing is on the order of 5-6 nm (50-60 A).For comparison, in Fig. 1C a strand prepared by the Klein-schmidt method is shown, which under the same conditions ofshadowing has a width of about 16 nm (160 A). This significantdifference in width is presumably due to the binding to DNAof cytochrome c, whose molecular weight (about 13,000) isabout 35 times greater than that of BAC (340/368).

In Fig. 1D and E partially denatured T7 DNA is shown.The regions previously considered to be "unmelted" in experi-ments with the cytochrome c method are seen to have aloosened structure with a foamy appearance, which is clearlywider [about 10 nm (100 A)] and less compact than nativeDNA (Fig. 1B). This phenomenon may be due to the unstack-ing of the bases prior to strand separation (19) and allows us torecognize local denaturation in earlier stages than is possiblewhen using cytochrome c.The appearance of single strands is strongly affected by

the presence of formamide or by additional staining, both ofwhich increase thickness and visibility (Fig. 1G to I), as alsoseen with the cytochrome c method (20). With the conven-tional shadowing used here, unstained single strands in thepresence of only 1.3% formamide (the amount carried overfrom the BAC stock) are just at the limit of the visibility(Fig. 1F). Their width is given by the size of the platinumgranules and is in the order of 2-3 nm (20-30 A), or just abouthalf of the width measured on double-strand preparations.Fig. 1K shows for comparison a strand prepared by thecytochrome c method with a width on the order of 8 nm (80A).The results of some contour length measurements are-sum-

marized in Fig. 2. Fig. 2A gives the length histogram ofdouble-stranded T7 DNA which was spread onto a hypophasecontaining 0.3 M\ ammonium acetate at pH 9. The meanlength (± standard deviation) of 155 randomly selected mole-

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86 Biochemistry: Vollenweider et al.

1.. I

/ /I (/'I,.

FIG. 3. (A) Single-stranded, denatured T7 DNA, conditions as in Fig. 1G. Magnification: X 35,000.(B) Partially denatured T7 DNA spread onto a hypophase containing 0.1 M ammonium acetate at pH 9 from a solution containing 1

pg/ml of DNA, 2.5 X 10-3% BAC, 3 mM phosphate buffer at pH 7.8, and 1.2% formaldehyde. The partial denaturation was achievedby heating the DNA in 0.01 M phosphate buffer (pH 7.8) and 3.5% formaldehyde for 10 min at 630. For contrast enhancement of thesingle-stranded regions, the grid was stained with uranyl acetate prior to carbon-platinum shadowing. Magnification: X46,000.

(C) E. coli RNA polymerase-T7 DNA complex. Polymerase (see arrow) per DNA mole ratio = 20. Complex formation essentially asdescribed in (12). The complex was spread onto a hypophase of 0.1 M ammonium acetate and 0.1% glutaraldehyde at pH 7.9 from asolution containing 1.4 pAg/ml of DNA, 2.5 X 10-3% BAC, 1.3% formamide, 0.1% glutaraldehyde, and 0.03 M triethanolamine at pH7.9. Magnification: X35,000.

cules was 11.8 ± 0.3 pum. Fig. 2B gives the size distribution ofdouble-stranded T7 DNA prepared by the droplet method.The mean contour length obtained under these conditionsfrom 109 randomly selected molecules was 12.54 + 0.24 pm.These contour lengths are compatible with values obtainedwith the intercalating dye method (11, 13) as well as reportedin the literature (21) after taking the influence of ionicstrength on the measured mass per unit length into account(22). Fig. 2C gives the histogram for 50 denatured T7 DNAmolecules prepared by spreading onto a hypophase of redis-tilled water. The hypophase method was used for the quan-titative analysis, because the presence of formamide sig-nificantly improved the visibility (Fig. 11), although higherproportions of obviously broken strands were present. For thestatistics every molecule that appeared to be unbroken bypure inspection was measured and included in the histogram.

Under these conditions, a mean contour length of 13.7 4 1.1pm was determined. This value is in reasonable agreementwith the accepted length for double-stranded T7 DNA; it is,however, very different from the 7.5 ± 0.4 plm reported (23)for single-stranded T7 DNA (ionic strength of the hypophase0.2 M).

Fig. 3A shows a denatured, single-stranded T7 DNA mole-cule as used for the histogram. Note the well unfolded andeasily measurable strand.

Fig. 3B shows a partially denatured T7 DNA molecule.One clearly recognizes double- and single-stranded regions.Also, extremely small loops of denatured areas are resolved(see also Fig. 1D).In Fig. 3C a complex between T7 DNA and E. coli RNA

polymerase is shown. The details of the complex formationwere essentially as described (13). Three RNA polymerase

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molecules are clearly visible near one end (see arrow!) of theT7 DNA molecule as was earlier demonstrated, using the in-tercalating dye method (13).

CONCLUSIONS

We have demonstrated that benzyldimethylalkylammoniumchloride as a spreading agent can fully replace cytochrome c

in the classical monolayer nucleic acid preparation methods.BAC has the advantage of being of low molecular weight(340/368) and, therefore, allows a better visualization of de-tails along nucleic acid molecules. It does not lead to a de-tectable thickening of the strands and, therefore, it is be-lieved that this method may be helpful for the study of nu-

cleic acid-protein interactions in single- as well as in double-stranded systems. Furthermore, it may be applied to theelucidation of secondary structures of various nucleic acidmolecules.

We thank Dr. R. Portmann for helpful and stimulating dis-cussions, and Mrs. H. Mayer-Rosa for excellent technical assis-tance. We thank also Drs. K. Muhlethaler, K. Downing, 0.

Kubler, and D. Turner for critically reading the manuscript.This work was supported by Schweizerischer Nationalfonds zurForderung der wissenschaftlichen Forschung, Grant no. 3,1590,73.

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