Article pubs.acs.org/biochemistry
Fluorescence Methods for Probing G‑Quadruplex Structure in Singleand Double-Stranded DNA Aaron J. Stevens,† Hannah L. Kennedy,†,‡ and Martin A. Kennedy*,† †
Department of Pathology, University of Otago, Christchurch, New Zealand Molecular Pathology Laboratory, Canterbury Health Laboratories, Canterbury District Health Board, Christchurch, New Zealand
‡
S Supporting Information *
ABSTRACT: Interest in exploring G-quadruplex (G4) structures in nucleic acids is growing as it becomes more widely recognized that these structures have many interesting biological roles and chemical properties. Probing the G4-forming potential of DNA with dimethyl sulfate, polymerase stop assays, or nuclease digestion are three commonly used techniques that usually employ radio-isotopic labels for visualization. However, as fluorescent labeling methods have grown in popularity and versatility, many laboratories have moved away from the routine use of radio-isotopic methods. We have adapted traditional procedures for structural analysis of G4-forming DNA sequences by using fluorescent labels and capillary electrophoresis and demonstrate their application to well-studied G4 structures, including c-MYC PU27 G4. The three fluorescent assays described here allow interrogation of G4 structures in double- and single-stranded DNA substrates, using either chemical or enzymatic cleavage. When combined, these techniques can provide valuable information for the investigation of G4 topology and structure, as well as visualizing any structural effects caused by interaction of quadruplexes with the complementary C-rich DNA strand. of fluorescent labels for nucleic acid analysis, many laboratories are no longer equipped to use radio-isotopic labeling. Fluorescence resonance energy transfer (FRET)22 is one of the few fluorescent methods available for analysis of G4 structure, but this is limited to investigating G4 in the context of single-stranded oligonucleotides. Indeed, most in vitro assays for structural analysis rely on single-stranded oligonucleotide templates and are not usually applied to probe G4 structure in the context of double-stranded DNA (dsDNA). Oligonucleotides do not represent the true genomic context of a G4, as they generally lack flanking regions that are present in genomic DNA, and this might have an impact on G4 stability and structure. Furthermore, the absence of a complementary DNA strand means that effects of Watson−Crick base pairing on formation of the G4 cannot be assessed. Therefore, methods that can analyze G4 structures in the context of longer, dsDNA molecules would be desirable. In this paper, we describe several fluorescent assays for probing G4 formation in both single- and double-stranded DNA, which are well suited to the genetics or biology laboratory. We used the same basic principles as traditional radio-isotopic analyses; however, the assays were adapted to utilize fluorescent markers that were incorporated into the DNA template in various ways. The fluorescent techniques described here have the advantage of allowing the simultaneous investigation of G4 structure on both strands of dsDNA
mong the five nucleoside bases commonly found in DNA and RNA, guanine is unique because of its ability to form Hoogsteen bonds at the N7 position of the pyrimidine ring. Four guanine nucleotides can form a square planar tetrad, stabilized by a central cation such as K+. When certain G-rich DNA sequences contain four repeats of two or more contiguous guanines separated by up to seven nucleotides, Gtetrads can form and stack into higher-order structures termed G-quadruplexes (G4). It is clear that there are many G4 motifs in the human genome, with initial bioinformatic estimates of ∼3000001 and more recent evidence of >700000 derived using an in vitro assay, based upon polymerase extension and nextgeneration sequencing.2 G4 structures have many proven and potential in vivo functions,3,4 including roles in telomere structure, DNA replication, gene regulation, transcription, and translation.5−7 After the first description of G4,8 most analyses were initiated by chemists with an interest in understanding and exploiting its structural diversity and properties.9,10 As the role of G4 in various diseases became evident, and with the growing recognition of the involvement of G4 in many areas of cellular and genome biology,11,12 increasing numbers of geneticists and cell biologists have become interested in analysis of G4. Traditional methods for G4 analysis such as dimethyl sulfate (DMS) footprinting 13−16 and DNA polymerase arrest assays16−19 are conducted with radio-isotopic labeling and thin layer polyacrylamide gel electrophoresis (PAGE).20,21 These methods are still widely used;20,21 however, because of the increasing level of sophistication, precision, and versatility
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© 2016 American Chemical Society
Received: April 7, 2016 Revised: June 1, 2016 Published: June 2, 2016 3714
DOI: 10.1021/acs.biochem.6b00327 Biochemistry 2016, 55, 3714−3725
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Figure 1. Outline of fluorescent methods. (A) Outline of the FOPE method, which was performed on either oligonucleotide or genomic templates. (B) Outline of FADFA and FANFA methods. Template 1 was a single-stranded oligonucleotide containing a 5′ FAM label. Template 2 consisted of short oligonucleotides fluorescently labeled with HEX (green) or FAM (blue) and used to label longer, complementary oligonucleotides through extension (gray) and ligation. The nonlabeled oligonucleotides (black) contained the G4 sequence of interest. Template 3 was double-stranded DNA generated using PCR, where differentially fluorescing primers are incorporated at opposing termini (HEX and FAM). Dashed gray lines indicate DNA polymerase extension.
average of at least three scans, obtained at 25 °C. An appropriate buffer blank correction was made for all spectra. Fluorescent Molecule Sizing by Automated Capillary Electrophoresis. Reaction products were prepared by addition of deionized formamide, followed by heating at 95 °C for 5 min to denature the DNA. Capillary electrophoresis was then performed on an AB 3130xl fragment analysis system equipped with a 50 cm capillary using a POP7 polymer and the size standard GS500LIZ. Data Analyses. Raw data were visualized with Applied Biosystems Peak Scanner version 2.0 and subsequently exported into Microsoft Excel (2007). The inferred nucleotide size of each peak was rounded to the nearest whole number, and peaks of a size smaller than or equal to that of the primer were removed as these were not relevant. The peak sizes were then manually plotted such that the x-axis represented the template DNA strand (DNA sequence or amplicon size) and the y-axis represented relative fluorescence units (RFU) for each peak height, which reflects the amount of primer incorporated into the products at each peak. To integrate experimental repetitions (minimum of three), the RFU was averaged at each nucleotide position, with error bars representing the standard error.
templates in one assay. For the validation of these assays, we focused on the well-studied PU27 region of the c-MYC oncogene;16,23−31 however, results were also validated against the commonly studied G4 motifs from the RET, VEGF, and BCL2 genes.32−34
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EXPERIMENTAL PROCEDURES Electrophoresis and DNA Recovery. Nondenaturing polyacrylamide gel electrophoresis (PAGE), agarose gel electrophoresis, and recovery of DNA from gels were performed as previously described.35 Band extraction and purification of polymerase chain reaction (PCR) products were performed using a MEGA-Quick Spin Kit (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do,Korea), according to the manufacturer’s recommendations. Circular Dichroism (CD) Spectroscopy. Oligonucleotides were purchased from Integrated DNA Technologies (IDT Pte. Ltd., Singapore). CD measurements and CD melting studies were performed on a Jasco J-815 CD spectrometer (Jasco Analytical Instruments), with a 1 mm path length quartz cuvette. The sample temperature was regulated with a Peltier controller. CD spectra were recorded from 340 to 200 nm in 1 nm increments, and the reported spectra correspond to the 3715
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Fluorescence Cleavage Footprinting Assays. Two fluorescence footprinting assays that used either DMS or Mung bean nuclease for cleavage were developed (Figure 1). We refer to these methods as the fluorescence analysis of dimethyl sulfate footprinting assay (FADFA) and the fluorescence analysis of nuclease footprinting assay (FANFA). The assays were performed on three DNA substrates, termed Templates 1−3 (Figure 1). The templates all contained fluorescent labels at the 5′ termini but were generated using different protocols, as described below. Template Design and Synthesis. Template 1 (Figure 1) was nonpurified 5′ FAM-labeled oligonucleotide (IDT) (Supplementary Table 1). The oligonucleotide length was restricted to a maximal length of 60 nucleotides by the manufacturer, and it was found that a minimum of 20 nucleotides separating the 5′ FAM label from the G4 motif was optimal for accurate fluorescent molecule sizing by automated capillary electrophoresis. Template 2 (Figure 1) was generated through ligation of four separate oligonucleotides [CmycPF1HEX, CmycPR1FAM, CmycFbindsR, and CMycRbindsF (Supplementary Table 1)] where CmycRbindsF contained the PU27 sequence. The 5′ flanking regions of CmycFbindsR and CmycRbindsF were complementary to differentially labeled fluorescent oligonucleotides [CmycPF1HEX and CmycPR1FAM (Supplementary Table 1)]. When the oligonucleotides were assembled into a double-stranded construct by annealing followed by ligation, the fluorescently labeled termini were located at the 5′ end of each strand (Figure 1). This approach created a double-stranded PU27 template, in which one strand was labeled with FAM and the other was labeled with HEX. This unique configuration allowed simultaneous interrogation of both strands in one assay. Annealing was performed by heating each oligonucleotide (0.5 μM) for 5 min at 95 °C in 200 μL of annealing buffer [10 mM Tris (pH 8.0) and 50 mM LiCl] and cooling the sample in a thermal cycler at a rate of 0.2 °C/min. Because of the larger size of the RET, VEGF, and BCL2 templates, gap filling was required to create a double-stranded template; this was performed using the non-strand-displacing T4 DNA polymerase, according to the manufacturer’s recommendations. The samples were then subjected to ligation by T4 DNA ligase (New England Biolabs Inc., Ipswich, MA) according to the manufacturer’s recommendations. To purify the desired template, the sample was concentrated by centrifugal evaporation, and full length templates were isolated by denaturing PAGE and visualized using SYBR Gold (ThermoFisher Scientific, Waltham, MA). Template 3 (Figure 1) was generated through PCR of genomic DNA with differentially labeled fluorescent primers (CmycPF1HEX and CmycPR1FAM) and involved two PCR amplification stages. Initial PCR amplification was conducted on genomic DNA as described below. Following agarose gel electrophoresis and UV visualization, the desired PCR band was excised and purified. The identity of each amplicon was verified using Sanger sequencing, and the remaining material served as a stock for reamplification prior to the FADFA. Reamplification was conducted using ∼1 pg of template DNA, according to the PCR cycling parameters mentioned below. The PCR product was then purified using an AcroPrep (PALL Corp., New York, NY) filter plate (omega 100K) and resuspended in 40 μL of Millipore water. To obtain sufficient quantities of DNA, the products of multiple PCRs were pooled.
Where both strands were labeled and bidirectional analysis was performed on dsDNA, the sizes of peaks originating from the reverse primer were subtracted from the overall amplicon length and plotted on the same axis as peaks generated by the forward primer. This allowed for graphical representation of fragments originating from each primer mapped to their appropriate position on the template sequence. Fluorescent Oligonucleotide Primer Extension (FOPE) Assays. These procedures were adapted from the radioisotopic DNA polymerase arrest method described by Weitzmann et al. 17 and where possible followed the experimental procedure for c-MYC PU27 outlined by Siddiqui-Jain et al.30 FOPE assays involved the extension of FAM-labeled oligonucleotide primers and were performed on single-stranded oligonucleotides and double-stranded genomic DNA templates (Figure 1). Each assay was conducted in triplicate and independently repeated three times (a total of nine assays per experiment). Negative control reaction mixtures for each assay contained template, primer, and all other reagents with the exception of Taq polymerase. FOPE Performed Using Single-Stranded Oligonucleotides. This approach (Figure 1) was performed using (nonfluorescent) synthetic oligonucleotides (IDT, Singapore) as templates, in a total reaction volume of 30 μL containing 1× PCR buffer with 1.5 mM MgCl2 (Roche Diagnostics), 0.25 μM FAM-labeled primer, 200 μM dNTPs containing 7-deaza-2′deoxyguanosine 5′-triphosphate (7-deaza-dGTP; Trilink Biotechnologies, San Diego, CA) in a 1:3 ratio with dGTP, 0.5 unit of non Hot-start Taq DNA polymerase (Roche Diagnostics), and 0.25 μM oligonucleotide. The addition of 7-deaza-dGTP was not crucial for polymerase arrest, but it was found to reduce the potential for structural interference by nascent PCR amplicons. Oligonucleotides, buffer, and nuclease-free water were heated at 95 °C for 3 min and then cooled to room temperature over 30 min. Taq DNA polymerase and dNTPS were then added to this mix at the indicated concentrations and incubated for 1 h at 55 °C. Reactions were terminated via the addition of 20% (v/v) stop buffer containing 1.5 M sodium acetate and 1.0 M βmercaptoethanol. One microliter of the sample reaction mixture was added to 9 μL of deionized formamide for sizing by capillary electrophoresis. FOPE Performed Using Genomic DNA. This approach (Figure 1) was performed in a total reaction volume of 30 μL containing 1× PCR buffer with 1.5 mM MgCl2 (Roche Diagnostics), fluorescently labeled primers (0.25 μM each), 200 μM dNTPs containing 7-deaza-dGTP in a 3:1 ratio with dGTP, 0.5 unit of Hot-start Taq DNA polymerase (Fisher), and ∼350 ng of genomic DNA. Cycling conditions consisted of an initial denaturation step of 95 °C for 2 min, followed by one cycle of 95 °C for 30 s, annealing ay 58 °C for 15 s, and extension at 72 °C for 1 min. Polymerase extension was terminated through the addition of phenol and chloroform and brief vortexing, after which the aqueous phase was removed and concentrated by centrifugal evaporation at 55 °C. DNA samples were then stored in a lyophilized state at −20 °C until capillary electrophoresis. For these experiments, the lyophilized DNA pellet was resuspended directly in deionized formamide. When using centrifugal evaporation to concentrate samples, salts from the PCR are also concentrated, which can interfere with fragment migration. To minimize this, sample volumes were limited to 30 μL. 3716
DOI: 10.1021/acs.biochem.6b00327 Biochemistry 2016, 55, 3714−3725
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Biochemistry Polymerase Chain Reaction Conditions. Unless stated otherwise, PCR amplification was conducted in a total reaction volume of 25 μL containing 1× PCR reaction buffer with 1.5 mM MgCl2 (Roche Diagnostics), each primer at 0.5 μM (IDT), each dNTP at 0.2 μM, 1 M betaine, 0.5 unit of Fisher Taq-ti polymerase (Fisher Biotec, Wembley, Australia), and ∼30 ng of human genomic DNA. Standard thermal cycling conditions consisted of an initial denaturation step of 95 °C for 2 min, followed by ≤35 cycles of 95 °C for 30 s, annealing for 57 °C for 15 s and 72 °C for 45 s, and a final extension of 72 °C for 5 min. DMS Treatment. Where specified, DMS was added to the reaction samples (generally 200 μL) at a final concentration of 1% and incubated at room temperature for 5 min. The reaction was quenched through the addition of sodium acetate (pH 7.0) to a final concentration of 0.3 M, along with 0.2 M βmercaptoethanol and 5 ng of salmon sperm. DNA was then precipitated by addition of an equal volume of isopropanol and stored overnight at −80 °C. After centrifugation at 14000g for 20 min, the DNA pellet was washed twice in 70% ethanol, resuspended in 50 μL of a 20% (v/v) piperidine/ethanol mixture, and incubated at 90 °C for 30 min. Piperidine was removed by centrifugal evaporation at 60 °C, and the sample was stored in a lyophilized state at −20 °C. Prior to capillary gel electrophoresis the sample was resuspended in deionized formamide. In later experiments, it was found that heating in deionized formamide at 95 °C for 30 min was sufficient to induce strand cleavage, circumventing the use of piperidine. FADFA Performed on PU27 Template 1. Nonpurified 5′ FAM-labeled oligonucleotide [termed Template 1 (Figure 1)] was reconstituted in Millipore water; 300 pmol of oligonucleotide was added to 9 μL of 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl or LiCl (negative control). Oligonucleotides were either heat denatured at 95 °C for 5 min and cooled at a rate of 0.2 °C/min or incubated at 37 °C for the desired period. One microliter of glycerol was added to the reaction mixture, prior to it being loaded onto a nondenaturing 12% polyacrylamide gel as previously described.35 Fluorescent oligonucleotide migration was monitored using direct visualization with a UV transilluminator (Alpha Innotech Corp.). After bands had migrated approximately two-thirds of the total length, the gel was removed from the apparatus and DNA bands were excised. The excised bands were added to 200 μL of appropriate G4 folding buffer [10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl], crushed, subjected to freezing− thawing cycles, and rotated overnight at 4 °C. Samples were then centrifuged at 14000g for 5 min, and the supernatant was removed using a pipet. Small polyacrylamide particles were removed by size exclusion centrifugation using a 100K AcroPrep 96-well filter plate. The filtrate was interrogated for G4 structural topology using CD spectroscopy. The sample concentration was then adjusted and subjected to DMS treatment. FADFA Performed on Template 2. To ensure that Template 2 did not contain G4 structure at the outset, 500 ng of the recovered sample was subjected to DMS analysis. To examine whether G4 can form on PU27 Template 2 without prior thermal denaturation of the double helix, 500 ng of the sample was suspended in 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl or LiCl (negative control). The sample was then incubated at 37 °C, consistent with the protocol published by Siddiqui-Jain et al.30 After incubation for the desired time course, the sample was subjected to DMS treatment. To
examine G4 formation of RET, VEGF, and BCL2 templates, samples were suspended in 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl or LiCl (negative control). G4 formation was examined after the samples were incubated for 5 min at 95 °C, followed by controlled cooling to room temperature at a rate of 0.2 °C/min. FADFA Performed on PU27 Template 3. Reamplified Template 3 (700 ng to 1 μg) was diluted in buffer at final concentrations of 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl or LiCl (negative control), and in a final volume of 200 μL. The sample was incubated for 5 min at 95 °C, followed by controlled cooling to room temperature at a rate of 0.2 °C/ min, to allow G4 formation. Samples were then treated with DMS. Using this technique, positions of cleavage could be resolved up to approximately 300 bp from each fluorescent primer. FANFA Performed on PU27 Templates 2 and 3. Templates 2 and 3 were interrogated using the single-strand specific endonuclease, mung bean nuclease (New England Biolabs Inc.). Samples of Template 2 (700 ng) or Template 3 (1 μg) were purified and resuspended in 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM KCl, according to prior materials and methods. To allow G4 formation, Template 2 was incubated at 37 °C for 24 h, without thermal denaturation. In an attempt to promote G4 formation in Template 3, the sample was incubated for 5 min at 95 °C, followed by controlled cooling to room temperature at a rate of 0.2 °C/min. Prepared templates were then incubated with 4 units of mung bean nuclease in appropriate buffer at 37 °C for 14 min in a reaction volume of 80 μL. This reaction was terminated via the addition of phenol and chloroform in a 1:1 ratio. After vortexing and centrifugation at 14000g for 5 min, the aqueous phase was removed and concentrated using a centrifugal evaporator. The lyophilized pellet was then resuspended in deionized formamide for size analysis by automated capillary electrophoresis.
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RESULTS AND DISCUSSION The range of investigations described below was performed using just three fluorescent oligonucleotides. We refer to these methods as fluorescent oligonucleotide primer extension (FOPE), the fluorescence analysis of dimethyl sulfate footprinting assay (FADFA), and the fluorescence analysis of nuclease footprinting assay (FANFA). FOPE is a technique that monitors in vitro replication by Taq DNA polymerase.17 When Taq polymerase encounters a stable G4 motif in the template DNA, amplification is hindered by the G4 structure, resulting in early strand termination.36 The FADFA and FANFA are both footprinting techniques for probing the structure of G4-forming motifs by assessing which guanine residues contribute to G4 formation. The FADFA relies on chemical modification and cleavage of the DNA template, whereas the FANFA relies on enzymatic cleavage. These three assays were applied to three DNA substrates, termed Templates 1−3 (Figure 1). The templates all contained fluorescent labels at the 5′ termini but were generated using different protocols. FOPE on a Single-Stranded Oligonucleotide Template. G4-induced arrest of Taq polymerase during PCR was investigated using a FOPE assay, modified from radio-isotopic and fluorescent methods.17,37,38 Validation of the method was achieved by comparison with published radio-isotopic polymerase arrest assay data for the well-characterized PU27 G4 motif (Table 1) of the human c-MYC gene region.30,39,40 3717
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27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1
The DNA sequence of PU27 (hg19 coordinates chr8:128,748,174−128,748,200) is presented in the top row in a 5′ to 3′ orientation where each G-tract is shown in bold (represented as G tracts 1−5 from left to right, respectively). Nucleotide numbering is presented in the bottom row, which corresponds to the nucleotides listed above, and is consistent with the numbering of PU27 used by SiddiquiJain et al.30
FOPE was performed on the 59-nucleotide oligonucleotide template (CMYCBindsF), which contained the PU27 G4forming motif flanked by five-nucleotide adenine repeats. Nucleotide numbering of PU27 in this paper uses the system of Siddiqui-Jain et al.30 (Table 1). Polymerase extension was initiated from the HEX-labeled primer CmycPf1HEX that bound 3′ of PU27 and encountered the G27 residue of PU27 as the first nucleotide. The arrangement of PU27 was consistent with genomic DNA and the 77-mer template used by SiddiquiJain et al. 30 and Yang and Hurley; 40 however, the oligonucleotide used in those studies had different flanking regions and primer binding sites. Polymerase arrest occurred at two dominant positions that corresponded to G23 and G20 of G tract 1, with minor termination also observed two bases prior at A25 (Table 1 and Figure 2). The remaining 5′ peaks represent extension of the full length template. These results are consistent with the findings of Yang and Hurley,40 who reported the primary position of arrest occurred at the four 3′ guanines, and Siddiqui-Jain et al.30 (in the supplementary data of their paper). This FOPE analysis of the single-stranded template therefore generated data analogous to those from traditional radioisotopic assays. FOPE of Genomic DNA Templates. FOPE was then performed directly on genomic DNA, where polymerase extension was initiated 22 bp 5′ of the PU27 motif. Use of a double-stranded template and two primers labeled with different fluorophores allowed the simultaneous, bidirectional, analysis of polymerase arrest sites on both DNA strands of genomic DNA, which would not be possible with radiolabeling. The input concentration of genomic DNA was a limiting reagent in this experiment, and therefore, the magnitude of the fluorescent signal was significantly reduced relative to that from analysis of oligonucleotide templates. We found the minimal amount of DNA per analysis to be ∼200 ng. Polymerase arrest on the G-rich strand of this doublestranded template corresponded to G18 (Figure 3), which was consistent with FOPE performed on single-stranded Template 1. An additional position of polymerase arrest was also observed that corresponded to a downstream G-rich motif at 88 nucleotides (not included in the oligonucleotide used for the previous experiment). Polymerase arrest at this position occurs within repeats of consecutive guanines that closely align to the sequence motif described by Belotserkovskii et al.24 During extension of the C-rich strand, two minor positions of polymerase arrest were observed. One of these positions occurred within the motif for PU27 at a position similar to that of arrest during extension of the G-rich strand. The second position of significant arrest occurred shortly after extension of the FAM-labeled primer (Figure 3). Comparison of FOPE performed on a synthetic oligonucleotide and genomic DNA demonstrated that additional polymerase arrest occurs at G-rich regions near PU27. Significant arrest occurred 18 nucleotides downstream of the PU27 motif, likely indicating that this region forms polymorphic structures. These additional arrest sites were not apparent when using the shorter oligonucleotide as a template, and this raises the question of whether investigation of G4 structure in oligonucleotide templates constrains G4 formation to an extent that limits the accuracy of any biological inferences. From this, it seems reasonable to suggest that traditional polymerase arrest assays, which only assess polymerase arrest using short oligonucleotide templates, likely represent an oversimplified scenario with less
a
T G G G A G G G G T
Table 1. Numbering of the PU27 G4 Motif Sequencea
G
G
G
G
A
G
G
G
T
G
G
G
G
A
A
G
G
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Figure 2. FOPE performed on the PU27 oligonucleotide. Single-stranded FOPE, performed on an oligonucleotide template representing PU27 (CMYCBindsF) and using non hot-start DNA polymerase. Template size (in nucleotides) is represented on the X-axis, and the corresponding base sequence is presented in the 3′ to 5′ orientation, as encountered by the polymerase during extension. Gray bars indicate the positions of polymerase arrest (six replicate assays), and the numbered peaks correspond to the nucleotide positions in the PU27 G4-forming motif. Relative fluorescence units (RFU) are illustrated on the Y-axis and are representative of the amount of primer incorporated into the termination products at each peak. The arrow represents the direction of primer extension.
Figure 3. Full length FOPE performed on genomic DNA. FOPE performed on genomic DNA, where peaks originating from polymerase arrest are represented on the X-axis, with nucleotide numbering indicated (from the 5′ end of the HEX-labeled primer). RFU are illustrated on the Y-axis and are representative of the amount of primer incorporated into the termination products at each peak. Dashed black lines connect positions of polymerase arrest with the corresponding colored nucleotide from the DNA sequence, where PU27 is underlined. Blue bars represent extension products initiated from the FAM-labeled primer and green bars products initiated from the Hex labeled primer. Arrows represent the direction of primer extension (green for HEX and blue for FAM). An equivalent polymerase minus negative control was performed that did not contain Taq polymerase, and no peaks larger than the primer size were observed (data not shown).
biological relevance than analysis of G4 in a wider genomic context, which is possible with the fluorescent capillary sequencing approach described here. FADFA Performed on PU27 Template 1. The fluorescence analysis of DMS footprinting assay (FADFA) was performed on the PU27 G4 region, and the results were comparable with published data from radio-isotopic DMS footprinting for this region.30 To isolate the G4-forming templates, the FAM-labeled oligonucleotide (CmycFAMG4) was incubated at 37 °C for the desired time and separated by nondenaturing PAGE (Figure 4). Four main bands were visible, where Bands 1 and 2 were previously determined to represent different G4 topologies.30 Bands 1−4 were extracted from the gel (Figure 4), purified, and separately analyzed by circular dichroism spectroscopy to verify G4 formation (Figure 5). Band 1 consistently failed to yield sufficient material for CD
analysis, although it was sufficient to perform FADFA. Upon CD analysis, Band 2 displayed an elliptical peak at 265 nm and a trough at 245 nm, indicative of parallel G4 formation, and consistent with published data.27,41 Band 3 displayed similar elliptical spectra; however, peaks were low and broad. Band 4 had an elliptical spectrum representative of a nonstructured oligonucleotide. G4-forming oligonucleotides extracted from Bands 1 and 2 were then interrogated using the FADFA. Band 1 DMS footprinting (FADFA) demonstrated cleavage protection at G tracts 1, 2, 4, and 5 (Table 1 and Figure 6A) in a pattern that was consistent with G4 formation. However, G20 and G23 from G tract 5 were cleaved, and guanine nucleotides 11−14 (G tract 3) were highly susceptible to cleavage, indicating an absence of Hoogsteen bonds and a lack of involvement of these regions in G4 formation (Figure 6A). The DMS footprint of Band 2 showed preferential cleavage at G 3719
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positions differed in the pattern of guanine cleavage indicates that FADFA nevertheless performed in a fashion very similar to that of traditional DMS analysis. FADFA Performed on PU27 Template 2. We next applied the FADFA to double-stranded oligonucleotides (Template 2) that were assembled by annealing and ligation and contained the PU27 motif on one strand. For this analysis, we also tested the hypothesis that G4 Watson−Crick basepaired (B-form) DNA could adopt G4 structure after the addition of KCl, without prior strand denaturation. The absence of G4 formation at the outset of the experiment was confirmed by performing the FADFA on the initial template in water, prior to the addition of KCl (data not shown). After the sample was subjected to G4 folding conditions (100 mM KCl, 37 °C, 48 h), the FADFA demonstrated clear protection from cleavage at G tracts 1, 2, 4, and 5, with cleavage occurring at G tract 3, G26 and G27 (Figure 7). This pattern of cleavage is consistent with that observed for Band 1 (Figure 6A) and indicates that in the presence of 100 mM KCl, short duplex DNA molecules underwent a transition to adopt G4 structure, without prior strand denaturation. The pattern of DMS footprinting in this assay was similar to that obtained by Sun and Hurley39 in negatively supercoiled dsDNA. The advantage of this assay is that the same fluorescent markers can be incorporated into different templates, permitting the simultaneous investigation of several G4 motifs. We therefore used this approach to examine three additional templates containing G4 motifs from the BCL2, RET, and VEGF genes (Figure 8). All samples prepared in the presence of 100 mM KCl demonstrated guanine protection in a pattern representative of G4 formation, reinforcing the applicability of these assays to other G4-forming motifs. FADFA Performed on PU27 Template 3. We next investigated the application of the FADFA to assess G4 structure in a 144 bp, fluorescently labeled PCR amplicon (Template 3) that contained the PU27 motif within the wider genomic context of the c-MYC gene (Figure 9). This approach allows the simultaneous interrogation of both DNA strands in a single reaction by using two different fluorophores; however, because the c-MYC template consisted of a single G4 and there was limited cleavage on the other strand, only data from the Grich strand are presented here. For this assay, we attempted to promote G4 formation by heat-denaturing Template 3 prior to analysis (Figure 9). When the assay was performed in KCl (Figure 9A), substantially less guanine cleavage was observed at G tracts 1−3 than when it was performed with LiCl (Figure 9B). The technique of DMS footprinting has several advantages and limitations.42 One significant limitation is that the pattern of a nonstructured template is very similar to that of a template that contains a diverse range of structures.43 This occurs because the footprint consists of a large number of cleavage sites, averaged over all template molecules in the solution. No obvious pattern of guanine protection was detected for Template 3 at the PU27 motif, which indicates reduced G4 potential or mixed structures are likely to exist in long dsDNA. This observation is supported by the work of Sun et al.,39 who described the requirement of negative superhelicity in duplex DNA for the formation of G4 at G tracts 2−5. This technique also has the limitation that each additional G4 motif requires the design of fluorescent primers for the specific amplification of that gene region. Additionally, because G4 structures are efficient inhibitors of Taq polymerase, a
Figure 4. Nondenaturing PAGE analysis of annealed c-MYC oligonucleotide. CmycFAMG4 (38 nucleotides) oligonucleotide preincubated in 100 mM KCl at 37 °C for 1 or 48 h, as indicated. The marker (M) was double-stranded Template 2 (78 bp). The minor product termed Band 1 was only faintly visible on the original gel.
Figure 5. CD performed on single-stranded PU27 Bands 2−4. CD spectroscopy performed on oligonucleotide bands excised from the gel presented in Figure 4. The long dashed line represents the CD spectrum of Band 2. The dashed−dotted line represents the CD spectrum of Band 3. The dotted line represents the CD spectrum of Band 4. Because of insufficient signal, a CD profile was not obtained from Band 1. Molar ellipticity (×105 deg cm2 dmol−1) is on the vertical axis and wavelength (nm) on the horizontal axis.
tract 1, G7, G16, G20, and G23. Additionally, G9 and G14 displayed an increased level of cleavage. Protection predominantly corresponded to G tracts 2−5, with the exception of G7 from G tract 2 and G16 from G tract 3 (Figure 6B). These DMS footprints were consistent with previous analysis using a radio-isotopic assay,30,31 except that G16 of Band 2 was preferentially cleaved over G14 (Figure 6B). Consistent with the CD analysis, Bands 3 and 4 did not present a pattern of guanine cleavage indicative of any structure (data not shown). Our findings reinforced prior observations that the length of the DNA template and the surrounding flanking sequences influence the structure and propensity of a motif to adopt G4 formation. The results obtained from single-stranded oligonucleotide templates were highly congruent with published data, with the exception of a consistent and unexpected lack of cleavage at G26 and G27, which was not documented by Siddiqui-Jain et al.30 Given the substantial differences between the traditional radio-isotopic methods and the methods developed here, the observation that only these two nucleotide 3720
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Figure 6. FADFA performed on c-MYC Template 1. FADFA performed on G4-forming CmycFAMG4 oligonucleotides, extracted from the gel presented in Figure 4: (A) Band 1 and (B) Band 2. The nucleotide sequence is on the horizontal axis where numbering corresponds to the original numbering of PU27.30 RFU are represented on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. Gray shading indicates G tracts 1−5 (from left to right, respectively).
Figure 7. FADFA performed on c-MYC Template 2. The nucleotide sequence of PU27 is on the horizontal axis, and RFU are on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. Because of the lack of guanine on the FAM-labeled strand, cleavage data for this strand are not presented in this experiment. Gray shading indicates G tracts 1−5 (from left to right, respectively).
than cleaving the DNA backbone (like DMS and S1 nuclease), this separately identifies the single-stranded portions of both DNA strands. We found that the fluorescence analysis of nuclease footprinting assay (FANFA) best detected G4 structure by visualization of the C-rich strand, which suggests that G4 formation may protect the G-rich strand to a degree from cleavage by mung bean nuclease. G4 formation in double-stranded DNA was mapped using enzymatic digestion, and the results were compared against the results of chemical DMS cleavage. The FANFA performed on Template 2 demonstrated two dominant positions of nuclease cleavage on the C-rich (HEX-labeled) strand (Figure 10). This indicates the presence of single-stranded DNA at positions that are complementary to G2 and G27 of the PU27 motif (Table
successful template may not be possible to obtain by PCR. We also found it necessary to visualize the untreated template to verify that short terminated products generated during PCR were not carried over. FANFA Performed on Template 2. Via a technique similar to DMS footprinting, single-strand specific endonucleases can be used to interrogate dsDNA molecules for regions of localized structure, which are recognized as ssDNA and cleaved.44,45 Dimethyl sulfate and piperidine are both hazardous chemicals that require substantial safety precautions and appropriate disposal. Nuclease digestion can provide a less noxious alternative; however, it does not provide the base pair resolution that can be obtained with DMS footprinting. Because mung bean nuclease nicks single DNA strands, rather 3721
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Figure 8. FADFA performed on additional G4 motifs. All assays were conducted on Template 2 preparations (Figure 1B). (A) Analysis of BCL2 G4 after heat denaturation in water (three replicate assays). (B) Analysis of BCL2 G4 after heat denaturation in 100 mM KCl (six replicate assays). (C) Analysis of RET G4 after heat denaturation in water (three replicate assays). (D) Analysis of RET G4 after heat denaturation in 100 mM KCl (six replicate assays). (E) Analysis of VEGF after heat denaturation in water (two replicate assays). (F) Analysis of VEGF after heat denaturation in 100 mM KCl (six replicate assays). The nucleotide sequence is on the horizontal axis for the G-rich strand in the 5′ to 3′ orientation. RFU are represented on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. Gray bars correspond to peaks originating from the FAM-labeled G-rich strand. Peaks originating from the HEX-labeled strand are not shown.
1). The FAM-labeled G4-forming strand was nicked at each terminal nucleotide of PU27, and internal nicks corresponded to the linking loops between G tracts 1 and 2 and G tracts 2 and 3 (Figure 10). Visualization was best for the C-rich strand, which under these conditions is likely to exist as nonstructured, single-stranded DNA, facilitating enzymatic cleavage. It is unclear why the relative fluorescence of each strand is not proportional; however, this could result from variability in key steps such as during ligation of the labeled oligonucleotides, or during the endonuclease digestion step. The FANFA performed on Template 2 complemented previous results, indicating G4 formation in the same regions identified by FADFA and FOPE analyses. FANFA Performed on PU27 Template 3. We next applied the FANFA to the investigation of the PU27 motif in
the 144 bp dsDNA template, which was generated by PCR (Template 3) (Figure 11). The FANFA demonstrated a prominent position of nuclease cleavage corresponding to G18 of the PU27 motif. Cleavage at this position was observed on the C-rich and G-rich template strands. Minor cleavage was also observed at two other G-rich motifs (85 and 107 nucleotides), which were consistent with the positions of polymerase arrest observed above (Figure 3). For this template, nuclease cleavage was observed only after denaturation and annealing in the appropriate buffer. Assays performed on Template 3 indicated the predominant position of strand cleavage was initiated part way through PU27 and extended into the 3′ flanking region (Figure 11). The positions of cleavage corresponded to positions of polymerase arrest observed during FOPE and positions of triplex formation 3722
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Figure 9. FADFA of c-MYC Template 3. The nucleotide sequence is on the horizontal axis for the G-rich strand in the 5′ to 3′ orientation, where PU27 is denoted by the black box. RFU are represented on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. (A) Analysis of Template 3 after heat denaturation in 100 mM KCl (six replicate assays). (B) Analysis of Template 3 after heat denaturation in LiCl (three replicate assays). Gray bars correspond to peaks originating from the FAM-labeled G-rich strand. Peaks originating from the HEX-labeled strand are not shown.
Figure 10. FANFA of c-MYC Template 2. The nucleotide sequence of PU27 is on the horizontal axis for the C-rich strand in the 5′ to 3′ orientation. RFU are represented on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. Green bars correspond to peaks originating from the HEX-labeled C-rich strand and blue bars to peaks originating from the FAM-labeled G-rich strand. Cleavage sites within PU27 are shown in bold. Results presented are the average of three independent reactions.
reported by Belotserkovskii et al.24 The detection of G4 formation by the FANFA appears to be contradictory to previous analysis using the FADFA (DMS footprinting), which failed to detect G4 formation in this template. This is likely to have resulted from the formation of polymorphic structures, reinforcing the conclusion that in dsDNA the c-MYC region is likely to contain a mixture of duplex DNA, G4 structures, and partially melted structures.39 This further highlights how analysis of synthetic oligonucleotides might provide an overly simplistic scenario of G4 formation.
foundation for both chemical and enzymatic analysis of DNA structure, as demonstrated by validation against published data for the PU27 motif of the NHE III regulatory region of the cMYC oncogene, and G4 motifs from the RET, BCL2, and VEGF genes (Figure 8). Substantial additional data were also obtained through these procedures that could not have been derived using existing techniques. Although this study has focused on the analysis of G4 DNA, with further optimization, it is likely that these techniques could be adapted to investigations of RNA, DNA binding proteins such as transcription factors, or other structures such as DNA triplexes. The use of fluorescent DNA visualization has several advantages over traditional methods, especially for a laboratory not equipped to handle radio-isotopes. Fragment sizing by automated capillary electrophoresis permits the rapid analysis
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CONCLUSION We have described three fluorescent approaches for the characterization of non B-DNA structure in both genomic and artificial templates. These assays provide a sound 3723
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Figure 11. FANFA of c-MYC Template 3. The nucleotide sequence is on the horizontal axis presented in the 5′ to 3′ orientation, and PU27 is underlined. RFU are represented on the vertical axis, where peak height corresponds to the proportion of molecules cleaved at that position. Green bars correspond to peaks originating from the HEX-labeled C-rich strand and blue bars to peaks originating from the FAM-labeled G-rich strand. Results presented are from a single reaction, repeated seven times.
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ACKNOWLEDGMENTS We acknowledge the support of the University of Otago Graduate Research Committee by providing a Postgraduate Publishing Bursary (Doctoral) to A.J.S. We thank Grant Pearce of the Biomolecular Interaction Centre, from the University of Canterbury, for access to the circular dichroism spectrometer. We also thank Allison Miller for constructive advice.
of large sample numbers, with a reduced level of exposure to radio-isotopes and acrylamide. Unlike radio-isotopic labels, fluorescent primers can retain activity for several years when they are stored in the absence of light, and dual fluorescent labels are easily used for simultaneous analysis of both DNA strands. Used together, FOPE, the FADFA, and the FANFA provide effective enzymatic and chemical tools for characterization of secondary structures in DNA. When combined, these protocols can provide valuable information about G4 topology and structure in dsDNA, as well as allowing visualization of any potential interaction due to the complementary strand. These techniques also allow for larger scale analysis of long DNA fragments, which contain multiple G4 motifs, where both strands can be simultaneously investigated.
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ABBREVIATIONS DMS, dimethyl sulfate; G4, G-quadruplex; RFU, relative fluorescence units; FOPE, fluorescent oligonucleotide primer extension; FADFA, fluorescence analysis of DMS footprinting assay; FANFA, fluorescence analysis of nuclease footprinting assay.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00327.
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REFERENCES
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Oligonucleotide sequences used during experimental procedures (Table S1) (PDF)
AUTHOR INFORMATION
Corresponding Author
*Department of Pathology, University of Otago, P.O. Box 4345, Christchurch, New Zealand. E-mail: martin.kennedy@ otago.ac.nz. Phone: +643 364 0530. Author Contributions
A.J.S. conceived and performed a majority of the experimental work and drafted the manuscript. H.L.K. contributed to the experimental work. M.A.K. contributed expertise and resources and edited the manuscript. Funding
This research was supported by the Marsden Fund (11-UOO175 BMS) Council from New Zealand Government funding, administered by the Royal Society of New Zealand, and by the Carney Centre for Pharmacogenomics, University of Otago. Notes
The authors declare no competing financial interest. 3724
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