The covalent attachment of multiple fluorophores to DNA containing

Attachment of Reporter and Conjugate Groups to DNA ... Qualitative and quantitative measurements of oligonucleotides in gene therapy: Part II in vivo ...
1 downloads 0 Views 1MB Size
Bioconjugate Chem. 1991, 2, 452-457

452

The Covalent Attachment of Multiple Fluorophores to DNA Containing Phosphorothioate Diesters Results in Highly Sensitive Detection of Single-Stranded DNA Nancy E. Conway and Larry W. McLaughlin’ Department of Chemistry, Boston College, 140 Commonwealth Avenue, Chestnut Hill, Massachusetts 02167. Received May 6,1991

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a “postassay” fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing >lo00 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.

INTRODUCTION

The detection of single-stranded DNA fragments resulting from procedures such as DNA sequencing has commonly relied upon the use of radioisotopes (32Pand 3 5 s ) in conjunction with autoradiography (I). Such radioisotopic techniques have become common for the visualization of the DNA fragments present in a sequencing ladder obtained after polyacrylamide gel electrophoresis. However, the use of radioisotopes is complicated by problems related to their storage and disposal as well as the health hazards arising from exposure to atomic emissions. Fluorescent, rather than radioisotopic, labeling techniques avoid these problems, but are themselves often limited by their sensitivity to detection (at least in the absence of sophisticated lasers, electronics, and computers). Many fluorescent labeling procedures reported to date have relied upon the introduction of a single fluorophore at one termini of the DNA fragment using a variety of chemical or enzymatic techniques (2). This approach is conceptually similar to radioisotopic end labeling, but typically requires the use of lasers and sophisticated electronic detection systems to locate the nucleic acid containing the fluorophore at femtomole or subfemtomole levels. Nevertheless, several DNA sequencing techniques have been developed that rely upon the detection of nucleic acid fragments carrying a single fluorophore (3-5). The incorporation of multiple fluorophores or other nonradioactive labels into nucleic acids has the potential to significantly enhance the detection limit for suitably labeled fragments by increasing the amount of signal present for a given quantity of nucleic acid. Two approaches are commonly used to incorporate multiple residues. (i) A modified nucleotide is synthesized that carries the fluorophore of interest or a functional group amenable to fluorescent labeling. Multiple fluorophores can be introduced into the DNA fragment as often as the modified nucleotide is inserted into the sequence. This has been very successful for biotin-based detection systems (6-8). (ii) An appropriate multifunctional linker is attached to the terminus of the nucleic acid to provide several sites for attachment of the fluorophores. Several related 1043- 180219 112902-0452$02.5010

studies have been recently reported (8-12). Both approaches will enhance the detection limit of the DNA, but are complicated by the necessity of synthesizing the modified nucleoside or multifunctional linker. Additionally, while enhanced detection sensitivity should be available with biological macromolecules containing multiple fluorophores, the expected increases in sensitivity can be complicated by quenching effects. In such cases, an increase in the number of fluorophores does not always result in a corresponding increase in measured fluorescence intensity (see, for example, ref 10). This observation suggests that nonradiative mechanisms for relaxation of the excited state can be present, or even predominate, with the close association of multiple fluorophores. Appropriate spacing of the fluorophores may reduce nonradiative relaxation processes and be advantageous in enhancing the sensitivity of detection. In the present paper, we examine an approach to the fluorescent labeling of nucleic acids in which the fluorophores are attached to the internucleotidic phosphorus residues of DNA fragments containing phosphorothioate diesters. This approach allows the incorporation of multiple fluorophores into the DNA, ideally one fluorophore for each nucleotide residue containing a phosphorothioate diester while spacing the fluorophores at roughly equal distances throughout the fragment. So that the covalent attachment of fluorophores does not alter the characteristics of the DNA, for example its electrophoretic mobility during assays such as polyacrylamide gel electrophoresis, the labels are introduced “postassay”-that is, after completion of electrophoretic analysis. In this procedure the DNA fragments are covalently labeled with the fluorophore while they remain embedded within the polyacrylamide gel matrix. EXPERIMENTAL PROCEDURES

Materials. Monobromobimane was obtained from Calbiochem (San Diego, CA) under the name Thiolyte reagent or from Molecular Probes (Eugene, OR). [ ( U - ~ ~ S I ~ A T P was obtained from New England Nuclear (Billerica,MA). M13mp18 DNA, DNA polymerase I, T4 DNA ligase, and the restriction endonucleases HpuII and A m 1 were products of New England Biolabs (Beverly, MA), while 0 1991 American Chemical Society

Bioconjugate Chem., Voi. 2, No. 6, 1991 453

Labeling of DNA wRh Multiple Fiuorophores

Sequenase was obtained from U.S.Biochemical Corporation (Cleveland, OH). The 2'-deoxynucleoside 5'-041thiotriphosphates) were obtained from Amersham (Arlington Heights, IL). Polyacrylamide gels were destained with a KS-10 shaker from BEA-Enprotech Corp. (Hyde Park, MA), and the fluorescent bands containing DNA were viewed on a transilluminator, Model TL-33 (Amm = 366 nm), manufactured by UVP, Inc. (San Gabriel, CA). Fluorescence in solution was measured with a PerkinElmer (Norwalk, CT) fluorescence spectrophotometer, Model 650-10s. The radioactivity was measured with an LKB Wallac RackBeta liquid scintillation counter (Uppsala, Sweden). Oligodeoxynucleotides were synthesized on an Applied Biosystems (Foster City, CA) 381A DNA synthesizer. Methods. Chemical D N A Synthesis. DNA fragments containing phosphorothioate diesters were prepared using phosphoramidite DNA synthesis techniques in which the iodine/water/THF/lutidine oxidation was replaced by sulfur/carbon disulfide/lutidine (13, 14). Using this approach, DNA could be prepared containing phosphorothioates at every internucleotidic site, or prepared as a mixture of derivatives with some sites containing phosphate diesters and others containing phosphorothioate diesters. Enzymatic D N A Synthesis. M13mp18 DNA was converted to the replicative form (RF) as follows. The template DNA (2.5 pg) and universal primer (0.1 pg) were annealed in 25 pL of buffer containing 100 mM NaC1,20 mM MgC12, and 100 mM Tris-HC1 (pH 8.0) by heating the mixture to 56 "C for 15 min followed by slow cooling to ambient temperature. The final 50-pL reaction containing dATP, dGTP, dCTP, dTTP (500 pM each), ATP (1mM), DNA polymerase I (Escherichia coli, 10 units), and T4 DNA ligase (10 units) was incubated overnight at 16 "C. Substitution of the appropriate dNTPaS derivativeb) for the corresponding dNTP(s) essentially as described by Taylor (16)allowed the enzymatic incorporation of phosphorothioate diesters in place of phosphodiesters. For internal standardization, [ ( u - ~ ~ S I ~ (1.4 A T Ci/mmol) P was employed in some experiments in the elongation reaction. Polyacrylamide Gel Electrophoresis. Gel electrophoresis was performed on 20 X 20 X 0.075 cm or 34 X 42 X 0.075 cm gels of 6% acrylamide, 0.6% bis(acrylamide), 3 mM NatEDTA, 7 M urea, and 50 mM Tris-borate (pH 8.3). Fluorescent Labeling of DNA Embedded within Polyacrylamide Gels. Postassay labeling was performed both in the presence and in the absence of 7 M urea. The DNA was fixed in the gel by soaking it in 10% aqueous acetic acid for 10 min. The gel was then transferred to a 4 mM solution of monobromobimane in 50% aqueous acetonitrile and allowed to react overnight (18 hours) in the dark. Visualization of Labeled DNA. The gel was removed from the monobromobimane solution and destained by shaking in 50% aqueous acetonitrile for 1-4 h. The destaining period appeared necessary because of minor reactions with the gel components and monobromobimane. Following a brief treatment (5 min) in 60% aqueous dimethylformamide, the DNA was viewed on a standard long-wavelength ultraviolet transilluminator at 366 nm. To quantify the results, the fluorescent bands of DNA were cut out of the gel and the amount of DNA present in the gel was determined by scintillation counting. Labeling and Isolation of a 15-mer Containing Three Phosphorothioate Diesters. After postassay fluorescent labeling (described above) with monobromobimane, the 5'-32Pend-labeled 15-mer was electroeluted for 2 h from a 20 PO polyacrylamide gel into dialysis tubing containing 0.5 X TBE buffer. The solution was evaporated to dryness, redissolved in 1 mL of distilled water, and desalted using

a column of Sephadex G-10. The DNA fragment was collected, evaporated to dryness, and redissolved in 3 mL of 5 mM KH2P04 (pH 4.5). The fluorescence of the solution was measured and the concentration of the 15mer determined by scintillation counting. The fluorescence as a function of concentration of the phosphorothioate diesters was plotted on the standard bimanelabeled Tp(s)T curve. Preparation of D N A Restriction Fragments Containing Phosphorothioate Diesters. M13mp18 DNA was converted to the RF form as described above. Substitution of the appropriate dNTPaS derivative(s) for the corresponding dNTP(s) allowed the enzymatic incorporation of phosphorothioate diesters in place of phosphodiesters. Restriction digests with AuaI were performed as follows. The AuaI reaction mixture contained RF M13mp18 DNA, 100 mM NaC1,20 mM MgC12, and 100 mM Tris-HC1 (pH 8.0). The reaction was initiated by the addition of the enzyme and incubated at 37 "C for 4 h. Generation of DNA Sequencing Ladders Containing PhosphorothioateDiesters. In a 15-pLmixture, M13mp18 single-stranded DNA (2.5 pg) and universal primer (60 ng) were annealed in buffer containing 40 mM Tris-HC1 (pH 7.5), 10 mM MgC12, and 50 mM NaCl by heating the mixture to 65 "C followed by slow cooling to ambient temperature. To the annealed mixture were added 2 ELLof the dCTP mix (2 p M each of dATPaS, dGTPaS, dTTPaS) and 6 units of Sequenase to give a total volume of 20 pL. The reaction was incubated for 10 min at 37 "C followed by the addition of 5 pL of A,G,C, or T mix that contained a final concentration of 300 pM dNTPaS and 3 pM ddNTP. After 10 min the reaction was stopped by the addition of 5 pL of formamide dye (deionized formamide, 20 mM NazEDTA, 0.03 % bromophenol blue, and 0.03 % xylene cyanol). RESULTS

We have previously described (13)the alkylation of phosphorothioate diesters as a simple method to introduce a large number of fluorophores into a DNA sequence already embedded within a gel matrix. In the present paper we have extended these studies to show that long fragments of DNA containing phosphorothioate diesters can be visualized at subfemtomole (