Electrochemical Platform.

Using a 15-pin setup, low-density DNA monolayers were formed on one set of electrode surfaces by DNA patterning from a secondary electrode. DNA substrates were optimized for length to balance the on–off signal differential with the ability of proteins to access the binding site; oligonucleotide lengths were tested with 10 nucleotides shorter and longer than the 26-mer used. For monolayer formation, thiol monolayers with 50% azide and 50% phosphate head groups first were prepared on the gold pins. We have previously characterized such low-density monolayers, and have found the total DNA coverage to be 20 pmol/cm² (34). Subsequently, specific DNA sequences were tethered to individual pins using electrochemically activated Cu¹⁺ click chemistry (29). The secondary electrode activates the inert copper catalyst precursor only at specific locations on the primary electrode surface. Multiple sequences of DNA with different methylation states in the restriction enzyme binding site were thereby patterned onto particular electrodes (Fig. 1). The multiplexed array allows five experimental conditions to be run in triplicate, enabling simultaneous detection from healthy tissue and tumor tissue along with a positive control.

Electrochemical measurements were obtained by constant potential amperometry over 90 s. Electrodes were measured after treatment with methyltransferase, either in its purified form or as a component of crude lysate, and again after treatment with 1,500 units/mL of the restriction enzyme BssHII. Lysate was prepared from cultured cells through a simple treatment of cell disruption followed by buffer exchange. Purified DNMT1 was first used to establish the sensitivity and selectivity of this platform (Fig. S1) and was subsequently included alongside lysate activity measurements as a positive control. The DNA-mediated signal remains protected from restriction, giving the same signal before and after enzyme treatment, or fully “on,” when the electrode is first treated with 65 nM DNMT1 protein on a hemimethylated DNA substrate in the presence of the SAM cofactor, although protein DNMT1 is easily detectable at a 15-nM concentration with 48 ± 3% signal protection after restriction. For 65 nM DNMT1 without SAM, only 33 ± 5% signal protection is observed. Similarly, little DNA protection (31 ± 6% signal protection) is observed with the unmethylated substrate. This is explained by the strong preference of DNMT1, as a maintenance methyltransferase, for a hemimethylated substrate.

Fig. 2 shows the raw data collected for two individual electrodes treated with crude lysate, one in which the signal is on in the presence of the SAM cofactor and one in which the signal is turned “off” in the absence of cofactor, given DNA restriction in the absence of methylation. Additionally, the reproducibility of the platform is shown (Fig. 2), with the 15 individual electrodes of a single assay. Interestingly, high concentrations of lysate were found to diminish the electrochemical signal, likely due to crowding on the DNA-modified electrode, limiting access and binding of the methyltransferase. Multiple concentrations of lysate were tested (Fig. S2); a concentration corresponding to 4,000 cells per electrode was dilute enough to allow access of DNMT1 to the DNA on the surface while still containing sufficient DNMT1 to produce measurable activity.

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Fig. 2.

Detection and reproducibility of DNMT1 activity in cell lysates using electrochemical platform. (Upper) Raw data from single electrodes in the presence or absence of SAM cofactor. In blue is the preliminary scan after an electrode modified with hemimethylated DNA has been treated with parent lysate, followed by treatment with 1 μM protease in phosphate buffer, both at 37 °C. In red is the scan after the electrode was treated with 1,500 units/mL BssHII for 1.5 h at 37 °C. (Upper Left) Final scan from an electrode with 160 μM SAM added to the lysate; the signals essentially overlay, indicating an “on” signal. (Upper Right) Final scan from an electrode without SAM added to the lysate; this produces an “off” signal. The constant potential amperometry was run for 90 s with an applied potential of 320 mV to the secondary electrode and −400 mV to the primary electrode. All scans were performed in Tris buffer (10 mM Tris, 100 mM KCl, 2.5 mM MgCl₂, 1 mM CaCl₂, pH 7.6) with 4 μM MB and 300 μM potassium ferricyanide. (Lower) Reproducibility within the two-electrode multiplexed array. Orientation of the tested conditions on the 5 × 3 array is shown (Inset) with circular electrodes colored to correspond with activity data represented in the bar graph. Both the electrodes treated with 65 nM purified DNMT1 on hemimethylated DNA with 160 μM SAM (green) and those treated with parent (WT) lysate in the presence of SAM (solid blue) show full signal protection. On electrodes without added SAM (blue striped and red striped) and on DNMT1 ^−/− lysate-treated electrodes in the presence of SAM (solid red), no signal protection is observed. Bar graph data are raw without standardization and error bars represent the SD over three measurements.

To further combat signal decreases caused by undesired DNA-binding proteins, after electrodes are treated with lysate, a protease treatment step is incorporated to remove remaining bound protein before the electrochemical measurements. This protease step further minimizes the possibility of remaining protein being bound either to the DNA or directly to the surface that could interfere with electrochemical measurements. Methyltransferase activity is then determined by the percent signal remaining after BssHII treatment. If the DNA is cut by the restriction enzyme, the signal is low, indicating little methyltransferase activity. It is noteworthy that the percent signal remaining is always nonzero because, even after restriction, a DNA fragment remains that can generate an electrochemical response with the noncovalent methylene blue redox probe; electrochemical amplification is proportional to the amount of bound methylene blue, and therefore to DNA length.

Differential Detection of DNMT1 Activity from Multiple Crude Cultured Cell Lysates.

We then tested the ability of the platform to differentiate between lysate from a parent (HCT116 wild-type) colorectal carcinoma cell line and a cell line that does not express DNMT1 (HCT116 DNMT1^−/−). As shown in Fig. 3, specific detection of DNMT1 activity is dependent on both the methylation state of the substrate and the presence of the cofactor SAM. The “signal-on” specificity for the hemimethylated DNA substrate indicates unambiguous DNMT1 activity (maintenance methylation), and not activity by other human methyltransferases, DNMT3a or DNMT3b, which do not show this substrate preference (de novo methylation) (7). Signal is dependent on the presence of DNMT1 (purified or from parent lysate) as well as the cofactor SAM (Fig. 3B) and the hemimethylated substrate (Fig. 3A). The remaining electrodes, treated either with parent lysate without SAM, or DNMT1^−/− lysate independent of the cofactor, had significantly attenuated signals after restriction enzyme treatment.

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Fig. 3.

Dependence of lysate activity on the DNA substrate and cofactor. The positive control is 65 nM purified DNMT1 on hemimethylated DNA with 160 μM SAM (green). All values are normalized to 100% protection of the purified DNMT1 electrodes. (Upper) Cultured cell lysate substrate (A) and cofactor (B) dependence. Electrodes treated with parent (WT) lysate on the hemimethylated substrate in the presence of 160 μM SAM (blue) showed full signal protection, whereas on an unmethylated substrate or in the absence of SAM (striped blue) no signal protection is observed. Independent of conditions, electrodes treated with DNMT1 ^−/− lysate (red solid and striped) showed no signal protection. (Lower) Biopsy tissue substrate (C) and cofactor (D) dependence. Electrodes treated with tumor lysate on the hemimethylated substrate in the presence of 160 μM SAM (blue) showed full signal protection, whereas on an unmethylated substrate or in the absence of SAM (striped blue) no signal protection is observed. Independent of conditions, electrodes treated with normal tissue lysate (red solid and striped) showed no signal protection. The data shown represent the aggregation of three independent replicate experiments, with three electrodes per condition per experiment.

Detection of DNMT1 Activity from Human Tumor Tissue.

Human biopsy tissue samples were similarly evaluated, and tumor tissue was readily distinguished from adjacent normal tissue (Fig. 3). Tissue biopsy samples were purchased from a commercial source and were thus handled and stored using conventional methods (snap freezing in liquid nitrogen after removal and storage at −80 °C for upward of 1 mo). The optimal amount of tissue for detection from these samples was found to be ∼500 μg per electrode; typical colon punch biopsies yield 350 mg of tissue (35). Samples of colorectal carcinoma tissue as well as the adjacent healthy tissue were prepared just as the cultured cell lysate, and showed differential activity with our electrochemical platform. The tumor sample, which showed greater signal protection, was sensitive both to substrate and to cofactor, consistent with high DNMT1 methyltransferase activity, similar to the cultured parent colorectal carcinoma cells. In contrast, the normal tissue sample showed low methyltransferase activity, as seen through the reduced electrochemical signal (Fig. 3). These data clearly indicate that tumors can be effectively differentiated from healthy tissue through electrochemical DNMT1 measurement with our platform. By Western blot, the relative abundance of DNMT1 in the tumor tissue compared with healthy tissue was quantitatively consistent with the electrochemical results (Fig. S3).

Lysate activities were also tested by a ³H-SAM assay, and relative activities of the various samples were comparable to those determined electrochemically (Fig. S4). However, as is typical for such radioactivity assays, activity measurements observed among trials of the ³H-SAM assay were extremely variable, much more so than with the electrochemical platform. Activity differences between the tumor and healthy tissue were seen only at concentrations of ∼1 mg of tissue per sample, significantly higher than what is needed for electrochemical detection. The time required to obtain the data for the ³H-SAM assay was additionally substantially longer.

Implications.

DNMT1 is an important clinical diagnostic target due to its connection to aberrant genomic methylation, which is linked to tumorigenesis. Direct detection of methyltransferase activity from crude tissue lysates provides an early method of cancer screening and can also inform treatment decisions. However, current approaches for detection of methyltransferase activity rely on radioactive or fluorescent labels, antibodies, and obtrusive instrumentation that limit their application in laboratories and clinics. Although electrochemical approaches generally overcome these limitations, direct detection of proteins from crude samples remains challenging because of the complexity of crude biological lysates, as well as the sensitivity required to analyze the limited material of small clinical biopsy samples.

Our electrochemical assay for DNMT1 methylation effectively circumvents these problems. Methylation is detected through the presence or absence of DNA surface restriction followed by electrocatalytic amplification. We avoid clogging the platform through the formation of low-density DNA monolayers, enabling target DNA-binding proteins in the lysate ample access to the individual DNA helices on the surface. Our platform is also sensitive and selective without the use of radioactivity, fluorescence, or antibodies through the combination of electrocatalytic signal amplification and the sensitivity of DNA CT chemistry to report changes to the integrity of the DNA. This allows for detection of DNMT1 from both cultured colorectal carcinoma cells and tissue biopsy specimens. No difficult or time-consuming purification steps are necessary, and, for each electrode, only ∼4,000 cultured cells or ∼500-μg tissue sample are required. Importantly, because of the multiplexed nature of this platform, we are able to assay for substrate specificity while simultaneously measuring normal tissue and tumor tissue lysates. Therefore, with our platform, healthy tissue is easily distinguished from tumor tissue using very small amounts of sample. More generally, this work may be applicable to sensing other DNA modifications and certainly should represent an important step in new electrochemical biosensing technologies.