Article pubs.acs.org/ac
Methylsorb: A Simple Method for Quantifying DNA Methylation Using DNA−Gold Affinity Interactions Abu Ali Ibn Sina,§,† Laura G. Carrascosa,*,§,† Ramkumar Palanisamy,§ Sakandar Rauf,§ Muhammad J. A. Shiddiky,*,§ and Matt Trau*,§,‡ §
Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Corner College and Cooper Roads (Building 75), Brisbane QLD 4072, Australia ‡ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland QLD 4072, Australia S Supporting Information *
ABSTRACT: The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as “Methylsorb”, which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold−DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.
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that the development of a simple, low cost, and robust detection method for DNA methylation is essential to enable the uptake and full utilization of such biomarkers in the clinic. More recently, Lin and Chang21 has reported an advanced technique, where the strength of the colloidal interaction between DNA and gold nanoparticles has been used to detect DNA methylation in cancer cell lines by naked-eye. This method avoids complicated surface modification procedures involved in many conventional methods. Herein, we have envisioned an extremely simple method referred to as “Methylsorb”, which is an effective and potential alternative to traditional methods and offers a label-free and real-time quantitative detection of DNA methylation. The underlying principle of Methylsorb involves the use of the sequence-dependent affinity interaction of DNA bases toward gold substrates. Since (i) bisulfite treatment converts methylation events into base changes and (ii) DNA base− gold affinity interaction (i.e., DNA adsorption) is highly sequence dependent,1,22−28 we hypothesized that the direct
eoxyribonucleic acid (DNA) methylation is a prevalent epigenetic alteration in cancer2 and, hence, considered one of the most promising biomarkers for cancer diagnosis, prognosis, and therapy monitoring.3,4 Early detection of aberrant DNA methylation offers a great potential to improve survival rates and decrease the cost of treatment by ameliorating prescription of ineffective therapies. DNA methylation strategies typically involve bisulfite5 modification of DNA to convert methylation events into base changes followed by comprehensive sequencing to read each individual base pair in the genomic region of interest.6−8 Over the past few years, much attention has also been focused on detecting DNA methylation from bisulfite treated DNA using sequencesensitive methods such as methylation specific PCR (MSP),9 methylation specific high resolution (MS-HR) melting,10 MALDI-TOF mass spectrometry,11 combined bisulfite restriction analysis (COBRA),12,13 hyperbranched rolling circle amplification (HRCA),14 or biosensing coupled with electrochemical15−17 or optical readouts.18−20 While most of these methodologies have significantly improved the analysis performance, their practical application is restricted due to the complicated surface and conjugation chemistries, multistep analysis procedures, or the use of labels. Therefore, we believe © 2014 American Chemical Society
Received: June 16, 2014 Accepted: September 16, 2014 Published: September 16, 2014 10179
dx.doi.org/10.1021/ac502214z | Anal. Chem. 2014, 86, 10179−10185
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Scheme 1. Methylsorb Approacha
a
Genomic DNA is derived from breast cancer cell lines. Bisulfite treatment and asymmetric PCR processing of these samples generate adenineenriched (blue) or guanine-enriched (red) antisense ss-amplicons for unmethylated and methylated templates, respectively. The ss-DNA amplicons are directly adsorbed on gold, and their adsorption is monitored in a real-time and label-free manner using surface Plasmon resonance readout. DNA adsorption generates an increase in the SPR signal with respect to the initial baseline, which is directly proportional to the amount of adsorbed DNA sequences on the SPR chip. (See also Supplementary Movie 1 of the Supporting Information).
methylated guanine-enriched sequences. The differential affinity interaction (i.e., differential adsorption) of adenine or guanine-enriched sequences, corresponding to the DNA basechanges associated with DNA methylation, can be determined in a single step via direct quantification of adsorbed DNA sequences in-real time. We used this approach to quantify the DNA methylation status of two CpG clusters located within the engrailed homeobox1 (EN1) and MIR200B gene promoter regions on DNA derived from MCF7 and MDA-MB-231 cells, respectively. As a standard reference for unmethylated DNA sample, we used whole genome amplified (WGA) DNA. To quantify adsorbed DNA sequences on gold chip, we used a surface Plasmon resonance (SPR) biosensing approach that detects changes in refractive index over time (i.e., changes in the SPR spectral shift) at the sensing surface, which is directly proportional to the relative mass increase associated with target adsorption, thus enabling the real-time and label-free monitoring of targets. This method has previously been used to detect regional DNA methylation using proteins with affinity to CpG rich regions30,31 or molecular inversion probes.18 Detecting DNA methylation through this gold-driven adsorption process in combination with SPR offers several critical improvements to conventional methylation technologies that include: (i) minimal sample and operational requirements, (ii) no use of surface-bound receptors and thus avoids modification procedures for the recognition layer of the SPR surface, and (iii) label-free and real-time monitoring that avoids any artifacts generally associated with the use of labels.
detection/quantification of adsorbed (via DNA−gold affinity interaction) bisulfite treated-DNA sequences could be an extremely simple way to analyze DNA methylation events. The schematic of the Methylsorb approach is illustrated in Scheme 1 (see also Supplementary Movie 1 of the Supporting Information). Initially, genomic DNA from cells was treated with sodium bisulfite to convert unmethylated cytosines into uracil, leaving methylated cytosines unchanged. Bisulfite converted samples were then amplified via a subsequent asymmetric PCR amplification step, which converts all uracils into thymines in the sense strand (i.e., the DNA strand running from 5′ to 3′ end) or into adenines in the antisense strand (i.e., the DNA strand running from 3′ to 5′ end). Because amplification is performed asymmetrically, it ensures that only ss-DNA sequences corresponding to the antisense strand are amplified. This step therefore introduces an accurate and unique way to create specific base changes on DNA, which ultimately can alter DNA−gold affinity interaction and thus give quantitative information on the original methylation status of the sample. This is because (i) it only generates ss-DNA, which is more prone than dsDNA to uncoil sufficiently to expose its bases and rapidly interact with the gold surface29 and (ii) it correlates a methylation profile with gold−DNA affinity behavior, as only the antisense strand, which becomes guanineenriched for methylated or adenine-enriched for unmetylated samples, is amplified. Since adenine bases are reported to have stronger affinity toward gold than guanine,1,22−28 the adenineenriched ssDNA sequences (i.e., unmethylated sequence) are expected to exhibit higher adsorption on gold than the 10180
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Figure 1. Detection of DNA methylation in synthetic samples within the selected EN1 region. (a) Nucleotide composition of oligo sequences. High and low affinity sequence motifs are noted (i.e., DNA sequence motifs of minimum 4 identical bases are underlined; sequence motifs corresponding to poly A/C mixed bases of minimum 4 nucleotides in length are highlighted in gray color). The A/G differences between the two strands are highlighted in red font. (b) SPR sensorgrams showing the spectral shift generated during the adsorption of methylated (red) and unmethylated (blue) synthetic EN1 region onto the gold chip. (c) Corresponding mean values of SPR spectral shift for methylated (M) and unmethylated (UM) regions. Each bar represents the average of three separate trials (n = 3), and error bars represent the standard deviation of measurements within experiments (relative standard deviation (%RSD) was found to be C ≥ G > T) of DNA onto gold surfaces. Notably, this outcome indicates that the Methylsorb approach can effectively distinguish methylated and unmethylated epigenotypes in synthetic samples even though they involved only 8 CpG sites (i.e., it has 8 A/G-base-changes “diluted” across 53 bases). Despite this high “dilution effect”, the difference between the magnitude of SPR signals for methylated and unmethylated epigenotypes was found to be significantly large (i.e., Δspectral shift ∼ 1.5 nm). It is also important to note that, for a given number of DNA bases, a DNA containing homopolymer (e.g., a sequence with clustered poly-A motifs) and a DNA containing more distributed DNA bases (e.g., adenine bases spread across the sequence) might essentially not exhibit the same adsorption behavior. We analyzed the sequence composition of the methylated and unmethylated epigenotypes and investigated the presence of any high gold-affinity motif containing (i) homopolymers (e.g., poly A) with a minimum of four bases and (ii) polymers enriched with a mixture of adenine and cytosine bases. The identified homopolymers (underlined) and polymers enriched with A and C nucleotide bases (highlighted in gray) are represented in Figure 1a. It is evident that the methylated and unmethylated DNA contains similar homopolymers both in numbers (e.g., 3 poly A and 1 poly-C motif) and in length. However, the polymers enriched with A and C bases were significantly larger in length for the unmethylated sequence. This is because bisulfite treatment and PCR processing of DNA not only lead to A/G-enrichment but also result in GC or AC enriched motifs in the methylated or unmethylated sequences (Scheme 1 and Figure 1a). Additionally, bisulfite conversion (followed by asymmetric PCR) of DNA also increases the adenine contents in the antisense strand for both epigenotypes. This enhances the possibility of forming larger AC motifs in the unmethylated sequences. We believe that this differential length of poly-AC motifs in methylated and unmethylated sequences is the key factor to offer a distinguishable SPR signal in our method. It is important to note that our adsorption (i.e., affinity) trend (A > C ≥ G > T) is different than the trend (G > A > C > T) previously reported on planar surfaces22 where DNA was adsorbed in aqueous condition but their thermal desorption energy trend was measured under ultrahigh vacuum conditions. Because the G > A > C > T trend was observed under a very different experimental condition, we believe that this might not be directly related to the adsorption (i.e., affinity) trend in aqueous conditions on planar gold surfaces. To investigate the applicability of this approach for numerous other CpG sites, we used BiQ analyzer software32 to predict the amplicon sequences that would result from bisulfite treatment and asymmetric PCR processing of the CpG rich regions from other cancer related genes. Table S2 (Supporting Information) represents the identified homopolymers (underlined) and polymers enriched with A and C nucleotide bases (highlighted in gray) in the methylated and unmethylated epigenotypes of five other cancer related genes. We observed that unmethylated
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RESULTS AND DISCUSSION To investigate the strategy of gold−DNA affinity interactions for detecting DNA methylation levels, we designed synthetic DNA oligos containing sequences (Figure 1a) similar to the bisulfite treated and asymmetric amplified sequences derived from the EN1 gene. The EN1 gene comprises a cluster of eight potentially methylated CpG sites within a span of 53 bases downstream to the transcription start site and identified as a potential biomarker in several cancer types.34−36 Samples containing synthetic methylated (200 nM) and/or unmethylated DNA oligos (200 nM) were driven through the SPR biosensor system using a syringe pump (see Materials and Methods). We initially performed our experiment at pH 3 because DNA adsorption extent and kinetics are reported to be facilitated at pH 3.37,38 However, we have observed that adsorption at pH 3 limits the ability of the Methylsorb approach to differentiate methylated and unmethylated targets (see Figure S1 of the Supporting Information). At low pH (i.e., pH = 3), adenines and cytosines become protonated, leading to high DNA adsorption compared to that under neutral pH conditions.37,38 Since both methylated and unmethylated sequences contain large numbers of cytosines and adenines bases, they could be strongly adsorbed on the gold substrate within a short period of time at pH A; red line) indicating the methylated status of this region in MCF7 cells. Responses were also very reproducible with a RSD value of less than 7% (n = 3) and were in agreement with our previous sequencing analysis data of this same region on MCF7 cells (see Supporting 10183
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Figure 3. Detection of DNA methylation in heterogeneous samples. (a) Representative sensorgrams showing the SPR spectral shift for the real-time adsorption of heterogeneous sample mixtures containing a designated percentage of DNA derived from MCF7 and WGA (i.e., MCF7/WGA DNA at 0%/100%, in blue; 25%/75%, in orange; 50%/50%, in purple; 75%/25%, in light blue; 100%/0%, in red). (b) Corresponding calibration plot. Each data point represents the average of three separate trials, and error bars represent the standard deviation of measurements within experiments.
Second, as Methylsorb is a receptor-free approach, it avoids complicated surface functionalization steps (i.e., attaching capture probe on the Au surface for target hybridizations, etc.). This also reduces variability associated with the surface modification process (i.e., it has high reproducibility) and makes the whole detection process relatively simple to accomplish. Third, since detection is always performed as a relative measurement by comparing adsorption data from target sample with respect to fully unmethylated internal standard (i.e., WGA derived amplicons in this case), it further provides a way for controlling intra and interassay variability. Moreover, due to the real-time nature of the Methylsorb approach, it offers in situ detection and absolute quantification in a single-step detection and does not require any complex data analysis. Finally, this assay could be addressed in a multiplex format using the SPR-imaging homologue.
particularly important at the early stages of cancer where tumor cells might be present in very low numbers. These factors contribute to generate heterogeneous DNA methylation. Because it has high relevance in cancer diagnosis, any new DNA methylation method should have the ability to detect heterogeneous DNA methylation at different levels. Therefore, we investigated the ability of our approach to detect heterogeneous DNA methylation (i.e., methylated targets in the presence of designated proportions of unmethylated DNA) by measuring the adsorption of DNA samples containing a mixture of methylated and unmethylated sequences. Samples were prepared by quantifying the copy numbers of MCF7 and WGA DNA followed by mixing designated proportions of methylated and unmethylated targets (i.e., MCF7/WGA DNA at 0%/100%, 25%/75%, 50%/50%, 75%/25%, and 100%/0%). Figure 3a represents the SPR sensograms of the heterogeneously methylated DNA samples. It was evident that the SPR signal was a function of methylation percentage in the heterogeneous mixture. It was also noted that there was a linear correlation (R2 = 0.9988) in signal enhancement with a decrease in methylation percentage (Figure 3b). This is presumably due to the increase in the adenine nucleotide content with an increase in the percentage of unmethylated DNA in the mixture resulting in an enhanced signal. Additionally, a significant difference in signal for samples containing 25% and 0% methylated targets (Figure 3b; 5.85 ± 0.07 versus 5.16 ± 0.11 nm) indicate that our approach was sensitive enough to detect at least 25% methylation changes. The level of sensitivity and excellent reproducibility demonstrated in this study indicates that our approach can potentially find its relevance in epigenetic investigations for heterogeneously methylated DNA samples. However, we believe that further optimization to the operating parameters (e.g., flow rate, buffer composition, and multiplexed SPR platform) can improve the detection sensitivity of our method. It is also notable that our method is not suffered by PCR bias (see Supporting Information). Although there are many conventional methods able to detect heterogeneous methylation at much lower levels (e.g., DNA sequencing6−8 or MS-HR melting 10), our method provides other advantages in comparison to the conventional methodologies. First, SPR is an extremely robust well-known technology for the quantitative analysis of molecular interactions,46 and since our customized SPR system18 is easy-to-build and relatively inexpensive, it represents a cost-effective alternative to existing costly methods such as RT-PCR, sequencing, or mass-spectrometeric readouts.
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CONCLUSION
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ASSOCIATED CONTENT
We have developed a simple, cost-effective, and real-time approach with the capacity to sensitively detect DNA methylation levels in genomic DNA samples. Our experimental findings demonstrate the accurate interrogation of CpG regions on multiple genes using an SPR biosensor and simultaneous validation using conventional bisulfite sequencing. This unique label-free and rapid (e.g., ∼40 min) approach based on the preferential nucleotide affinity toward gold can effectively detect DNA methylation levels, thereby eliminating the need for sequencing approaches. We have demonstrated the feasibility of using this approach to sensitively (e.g., 25% methylated target) and specifically detect methylated targets in the presence of a large excess of unmethylated DNA. This method also has the potential to detect global hypomethylation, one of the most important biomarkers of cancer, since the large cohort of CpG regions involved in this type of analysis might promote a large difference in the adsorption profiles of normal and hypomethylated samples. We envisage that this simple and rapid approach can potentially find its relevance in diagnostic settings.
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org 10184
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. Tel: +61-7-33464178. Fax: +61-7-33463973. *E-mail:
[email protected]. Tel: +61-7-33464178. Fax: +61-7-33463973. *E-mail:
[email protected]. Tel: +61-7-33464178. Fax: +61-733463973. Author Contributions †
A.A.I.S. and L.G.C. contributed equally. L.G.C., M.J.A.S., and M.T. designed the experiments and supervised the project. A.A.I.S., L.G.C., and R.P. conducted most of the experiments. S.R. designed and fabricated the SPR sensor chips. All authors discussed the data and wrote the paper. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge funding received by our laboratory from the National Breast Cancer Foundation of Australia (CG12- 07). This work was also supported by the UQ fellowship (2012001456) and ARC DECRA and DP (DE120102503, DP140104006).
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