DALE ,\ND
W A R D
Mercurated Polynucleotides: New Probes for Hybridization and Selective Polymer Fractionation? Roderic M. K . Dale* and David C. Ward
ABSTRACT: Polynucleotides containing covalently bound mercury atoms have been prepered by chemical or enzymatic syntheses and some of their physical and biochemical properties studied. The mercury substituents do not appear to alter significantfy normal polynucleotide structure. Mercurated polymers function efficiently as templates for nucleic acid polymerases, they are fully susceptible to degradation by standard nucleases, and their denaturation and reannealing properties resemble those of the corresponding nonmercurated polymers. While the Tm’sof D N A duplexes are lowered by extensive mercuration, the T,’s of DNAR N A hybrids and R N A duplexes are either unaffected or elevated. Mercuration, as would be expected, greatly increases the buoyant density of both DNA and R N A . The introduction of as few as one mercury atom per 200 bases
permits the selective and quantitative retention of the mercurated polymer probe (and associated nucleotide sequences) on columns of sulfhydryl-agarose. The use of mercurated nucleotides (as polymerase substrates) and oligonucleotides (as primers) in conjunction with sulfhydryl-agarose chromatography provides a simple and efficient method for the isolation of selected polynucleotide sequences, such as specific in vitro transcription products or terminal fragments of duplex DNA. Products adsorbed to the affinity resin are readily recovered for further analysis by eluting with buffers containing mercaptoethanol. Although the mercury-carbon bond is somewhat thermolabile, mercurated polynucleotides are suitable as probes in low temperature hybridization studies.
S p e c i f i c nucleotide sequences and genome fragments are currently being used to probe the structural organization of complex nucleic acids. This approach would be augmented considerably by a simple procedure for selectively recovering such probes and their associated “target” sequences. W e have previously described (Dale et al.. 1973. 1975) procedures for the introduction of covalently bound mercury atoms into polynucleotides. Mercurinucleotides, being organomercurials, exhibit an extremely high affinity for mercaptans. Polynucleotide sequences containing even a low level of mercury substitution should, therefore, be readily separated from unmodified nucleic acids by chromatography on resins containing sulfhydryl groups. The utility of the mercury atom as a molecular “tag” for sequence or hybridization probes depends upon a number of factors: ( I ) the mercury atoms must neither seriously perturb the structure (or biochemical properties) of polymer duplexes nor interfere with the annealing of single strands; (2) the mercury-carbon bond must be reasonably stable; and (3) the in vitro fidelity of mercurinucleotides as polymerase substrates should be equivalent to that of their natural counterparts. Examination of the chemical, physical, and biochemical properties of mercurated polynucleotides shows that these criteria can be satisfied. In addition, the results described here demonstrate that mercurated and nonmercurated nucleotide sequences can be rapidly and selectively resolved by chromatography on sulfhydryl-agarose.
scribed (Dale et al., 1975). Standard nonradioactive nucleotides were obtained from P-L Laboratories and Sigma Biochemicals. Radioactive nucleotides and [*03Hg] mercuric acetate were products of New England Nuclear Corporation. [35S]Mercaptoethan~l was obtained from Amersham Radiochemicals; [‘4C]cysteine was from Schwarz/Mann. Escherichia coli D N A polymerase I (fraction VII) was purified by the method of Jovin et al. (1969). E . coli R N A polymerase was prepared and assayed as described by Burgess (1969). Purified T 4 D N A polymerase and E . coli exonuclease I11 were the generous gifts of Drs. P. Englund and L. Enquist, respectively. Pancreatic DNase, pancreatic RNase, RNase T I , RNase T2, micrococcal nuclease, and spleen and venom phosphodiesterases were obtained from Worthington and Sigma. Calf thymus DNA (Grade V ) , poly (A), and poly(U) were purchased from Sigma; poly(dT) was from Miles Laboratories and bulk E . coli t R N A was from Schwarz/Mann. Yeast phenylalanyl-tRNA was the kind gift of Dr. A. Rich. Synthetic polydeoxynucleotides were prepared according to published procedures: poly[d(A-T)], Richardson et al. ( 1 964); poly(dG)-poly(dC), Radding et al. (1962). Ultraviolet absorption spectra were recorded using a Cary 15 or a Beckman 25k spectrophotometer. Thermal denaturation profiles were done in water-jacketed cuvets. The temperature of the circulating water was controlled using a Lauda K4R (or Haake FE) circulating water bath: temperature changes were programmed a t approximately 1 . O o / min. The temperature in the sample cuvets was measured using a Yellow Springs 42SC thermister probe. Absorption and thermal changes were continuously monitored on two synchronized recorders. Large-plaque SV40 virus h a s used to infect confluent monolayers of secondary African green monkey kidney cells at an input multiplicity of 2-5 plaque forming units/cell. Tritiated thymidine was added 24 hr post infection (10 pCi/
Materials and Methods The 5’-triphosphates of 5-mercuriuridine and 5-mercurideoxyuridine were prepared and purified as previously de_ _ _ ~-____-~ From the Department of Molecular Biophysics and Biochemistrq, Yale University School of Medicine, N e w Haven, Connecticut 06510. Received December 6, 1974. This work was supported by National Institutes of Health Grants GM-20230-02, GM-20124-02. a n d C A 16038-01.
MERCURATED POLYNUCLEOTIDES
ml, 5 X M ) and progeny virus isolated 3-4 days later. The virions were purified by isopycnic centrifugation in CsCl and the viral D N A was released by treatment at 50' with 1% sodium dodecyl sulfate-1 m M EDTA (Trilling and Axelrod, 1970). Form I and form I1 SV40 D N A was prepared by isopycnic centrifugation in CsCl containing ethidium bromide (Radloff et al., 1967). SV40 C R N A was prepared from form I SV40 DNA as described by Westphal (1970). The reaction mixture (2.0 ml) contained: Tris-HCI (pH 7.8), 0.05 M; MgC12, 4 mM; MnC12, 1 mM; CTP, GTP, UTP, and [I4C]ATP (3 X lo3 cpm/nmol), 1 m M each; 2-mercaptoethanol, 5 mM; form I SV40 DNA, 45 pg; R N A polymerase, 20 units. The reaction was incubated at 37' for 1 hr. Pancreatic DNase was added to a final concentration of 50 pg/ml and the mixture incubated at room temperature for an additional 20 min. The solution was extracted twice with an equal volume of phenol and the R N A in the aqueous phase precipitated by the addition of 3 volumes of ethanol. After standing at -20' overnight the solution was centrifuged for 15 min at 27,OOOg and the R N A pellet dissolved in 0.5 ml of 0.01 M Tris-HC1 buffer (pH 7.4). The sample was dialyzed for 24 hr against two 1-1. changes of 0.05 M sodium acetate buffer (pH 6.0). 60 pg of [i4C]SV40 C R N A (1.9 X lo3 cpmlpg) was recovered after dialysis. ['4C]SV40 C R N A was mercurated as follows: [203Hg]mercuric acetate (1.1 X lo4 cpm/nmol) was added at a final concentration of 2 X M to a 0.5-ml solution of SV40 C R N A (24 pg) in 0.05 M sodium acetate buffer ( p H 6.0). This solution was heated, in parallel with an identical sample lacking mercuric acetate, for 30 min at 50'. The reactions were terminated by the addition of 0.2 ml of quench buffer (0.01 M Tris-HC1 (pH 7.5)-1.0 M NaCl0.1 M EDTA) and the samples dialyzed against three 1-1. changes of T N E buffer (0.01 M Tris-HC1 (pH 7.5)-0.02 M NaC1-0.001 M EDTA). The second dialysis solution also contained M mercaptoethanol to provide an appropriate mercury ligand for hybridization. As determined from the 203HgCpm/OD260 ratio, 3.2% of the total bases in the SV40 C R N A was mercurated. Sulfhydryl-agarose was prepared following the procedure of Cuatrecasas (1970). Both Sepharose 6B (Pharmacia) and Bio-Gel A5M (Bio-Rad) have been used as the basic resin support with equally satisfactory results; 100 g of resin, after extensive washing, was suspended in an equal volume of distilled water. Finely powdered cyanagen bromide (250 mg/ml of packed resin) was added to the stirred slurry in a well-ventilated hood. The pH was maintained as near as possible to pH 1 1 by the dropwise addition of 10 N N a O H while the temperature was held at 20' by the addition of crushed ice. After proton release had subsided (1 530 min), the suspension was quickly transferred to a course sintered glass funnel and washed under suction with 1500 ml of water (adjusted to pH 10 with sodium hydroxide); 100 ml of a 2 M solution of ethylenediamine (adjusted to pH 10 with 6 N HCI) was added immediately to the washed beads and the reaction allowed to proceed, with continual stirring, for 20 hr at 4'. The resin was then washed with 4 1. of water and suspended in 100 ml of 1.0 M NaHC03, adjusted to pH 9.7; 16 g of N-acetylhomocysteine thiolactone was added and the solution, which had a pinkish hue, was stirred at 4' for an additional 24 hr. Since incomplete substitution of the aminoethyl-agarose intermediate could possibly elevate the level of nonspecific resin adsorption (see text), a large excess of N-acetylhomocysteine thiolactone
Time (hours) FIGURE 1: Time course of poly[d(A-T)] (0) and poly[d(A-HgU)] ( 0 )synthesis with E. coli DNA polymerase. I. Reactions (1 .O mi) con-
tained Tris-HCI buffer (pH 7.4). 50 mM; MgC12, 10 mM; [3H]dATP (4.2 X lo3 cpm/nmol), 1 mM; T T P or Hg-dUTP, 1 mM; 2-mercaptoethanol, 10 mM; poly[d(A-T)], 0.07 mM; and 3 units of DNA polymerase I (Jovin et al., 1969). The reactions were incubated at 37O. At the indicated times 50-11 aliquots were removed and 4 ml of cold CI3CCOOH solution (100% C13CCOOH, saturated sodium pyrophosphate, saturated trisodium phosphate, and water, 1:1:1:7) was added and the samples, after standing for 10 min on ice, were filtered onto Whatman G F / A filters. The filters were washed with 50 ml of C13CCOOH solution, dried, and counted in a Packard scintillation counter.
was used in these reactions. The sulfhydryl-agarose product was washed extensively with 6-8 1. of 0.1 M NaCl containing 1 m M mercaptoethanol followed by 1-2 1. of 0.1 M NaC1. The degree of sulfhydryl substitution was determined spectrophotometrically using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman, 1959) and by quantitating the binding of [203Hg]mercuric acetate. The sulfhydryl content, which varied from preparation to preparation, ranged from 1.0 to 8.8 pmol/ml of resin. The trinitrobenzenesulfonate color test, described by Cuatrecasas (1970), confirmed that in all cases complete amino group substitution had occurred. Results The mercurated analogs of the alternating copolymers poly[d(A-T)] and poly[r(A-U)] were studied in some detail because of their structural simplicity and their facile enzymatic synthesis. As shown in Figures 1 and 2, when poly[d(A-T)] is used as a template, dUTP-HgSE' and UTPHgSE are readily polymerized by E . coli D N A and R N A polymerases, respectively. The incorporation of the mercurinucleotides is totally dependent on the presence of a mercaptan (or cyanide ion) to act as a mercury ligand. The chloride or acetate salts of Hg-dUTP or Hg-UTP are not polymerized (Figure 2, Dale et al., 1973) and completely inactivate R N A polymerase, an enzyme sensitive to thiol modification. The Hg-S bond is essentially covalent, with an association constant of about loi6. in contrast to the
'
Abbreviations used are: Hg-U, 5-mercuriuracil; Hg-C, 5-mercuricytosine; UTP-HgSE and dUTP-HgSE, the 5-mercuri derivatives of UTP and dUTP in which the mercury ligand is 2-mercaptoethanol; Hg-UTP and Hg-dUTP, the 5-mercuri derivatives of UTP and dUTP in which the mercury counterion is chloride, acetate, or bicarbonate; poly(Hg-U), the homopolymer of 5-mercuriuridine; poly[d(A-HgU)], an alternating copolymer of deoxyadenosine and S-mercurideoxyuridine; poly[r(A-U)], an alternating copolymer of adenosine and 5-mercuriuridine. Unless otherwise specified the mercury ligand in poly[d(AHgU)] and poly[r(A-HgU)] is mercaptoethanol. BIOCHEMISTRY, VOL.
14,
NO.
t t , 1975
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DALF
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Table I : The Polymerization of ['3]Mercaptoethanol and [I4C]Cysteine by E. coli DNA Polymerase 1 when Hg-dUTP is used as a Substratc.0 Nanomoles of Radiolabel Polymerized Expt No.
1 2 3 4 5 6
Substrates [ 'HI dATP + [ *"'Hg] dUTP-HgSE [ 'HI dATP + dUTP
['HI ['HI [ 'H] [ 'HI
['3]Mercapto-
Radioactive Mercaptan
[ 'HI dAMP
4.70 4.85 4.55 1.53 16.2 1.49
Mercaptoe thanol Mercaptoethanol
dATP + Hg-dUTP dATP + [ ''Hg] dUTP-HgCys dATP + dUTP dATP + Hg-dUTP
['O'HgldUMP
ethanol
[ 14C]Cysteine
__.__
4.65
