Using as little as 1 picogram of purified DNA sample (think 2.5 trillion times lighter than a penny), scientists at the University of Brisbane have developed a method that permits the swift detection of cancer DNA in a patient sample of cell-free DNA, which circulates systemically. These researchers took advantage of the finding that cancers have drastically different patterns of methylation on their genomic sequences than normal cells. These endow the DNA with unique physical properties, including the way in which it can bind to gold nanoparticles. Using this system, a color-change of a mixture comprised of DNA, gold nanoparticles, and a salt solution is the readout for presence or absence of methylation patterns that are indicative of cancerous DNA.
As the medical community rapidly embraces the paradigm of precision medicine to guide specific treatment modalities, there is increasing pressure to distinguish a cancerous cell, protein, or DNA molecule from a normal one.
Gaining notoriety in the field of cancer biology is investigation into epigenetics, or processes that regulate our genes, their expression, and mediate their downstream effects. A key element of this field is the process of DNA methylation, in which a DNA methyltransferase catalyzes the addition of a methyl group to a cytosine nucleotide, generating 5-methylcytosine.
It has been long known that cancerous DNA has perturbed methylation patterns in comparison to bulk normal cells, at both the regional and global levels.
The authors coin this genome-wide distribution of methylation marks as the cell’s Methylscape, and its unique physical properties are proving to be more helpful than one might initially suppose.
Methylation events are largely restricted to genomic regions known as CpG islands, which are comprised of a high proportion of C and G nucleotides, and are implicated in genetic regulation.
In the normal genome, CpG islands are methylated in the intragenic regions (in between gene bodies), while the CpG sites around gene promoters are depleted of methylation.
The cancer context flips this trend, and we observe a loss of methylation in the intragenic regions, accompanied by a gain in clustered methylations at surrounding promoter regions, resulting in a net hypomethylation state, as shown in the image below.
Left: differences between the cytosine and methylcytosine nucleotide, which is the major species of DNA methylation. Right: methylation patterns are different between normal and cancerous genomes, with evenly distributed mC sites in the former, and clustered, localized methylation hotspots in the latter. Image credit: Sina et al, doi: 10.1038/s41467-018-07214-w.
Methylation profiling has even been utilized to identify many types of cancer from their normal pairs, but these involve extensive laboratory techniques such as bisulfite sequencing that are burdensome, time intensive, and prone to error.
Biophysical studies have been analyzed in basic biology contexts to predict how DNA methylation can have effects on the physical properties of DNA structures in vivo, particularly in regards to nucleosome assembly.
Researchers at the Center for Personalized Nanomedicine out of the University of Brisbane in Australia decided to combine these approaches of Methylscape-informed cancer identification with the unique physical properties of methylated-DNA to make a broadly applicable platform for cancer discrimination.
Based on their microscopy data of DNA of cell lines with fixed amounts of DNA methylation, they observed that the self-assembly of DNA complexes was directly correlated with global methylation levels.
These altered kinetics of self-assembly endow the DNA molecules with different properties of adsorption, or physical binding, onto metal surfaces.
The authors chose gold (Au2+) for this surface, due to the large volume of literature in the field that uses gold nanoparticles.
They found that the capacity of the DNA to bind a solid gold electrode plate was parabolically correlated with global DNA methylation (shown below).
Left: proportion of global genome methylation as well as its sub-patterning relegate its ability to adsorb. Right: the different physics of DNA folding with and without cancer-associated methylation patterns affects adsorption onto gold surface. Image credit: Sina et al, doi: 10.1038/s41467-018-07214-w.
The workflow of their proposed model is as described as follows and illustrated in the image below.
Genomic DNA from a patient is purified and added onto a gold electrode surface for a brief adsorption incubation. Differential Pulse Voltammetry (DPV) is measured and compared between the experimental sample, and a DNA-free baseline control.
This serves to measure adsorption efficiency, which is a surrogate marker for the proportion of the DNA that is physically available to bind the gold surface.
The fully methylated and fully unmethylated cell lines had the lowest DPV perturbation measured, followed by the normal control Human Mammalian Epithelial Cells (HMEC).
Both cancer cell lines used in the experiment exhibiting the greatest DPV change.
Experimental workflow to quantitatively measure DNA adsorption onto gold electrodes. The standardized output is the change in DPV following adsorption. Image credit: Sina et al, doi: 10.1038/s41467-018-07214-w.
While this protocol is quantitatively thorough, the authors sought to ease broad application, and further simplified the process by turning to colloidal gold nanoparticles (AuNPs).
Upon the addition of salt, SSC 5X, to a solution of AuNPs, the nanoparticles quickly aggregate and precipitate out of solution to cause a clear pink → blue color change.
If the AuNPs are prevented from aggregating due to previous binding to another substrate, such as DNA, then the pink color will persist.
This method (illustrated below) is quite sensitive, and requires only 1 picogram of cell-free DNA (cfDNA), which is readily derived from patient plasma.
Purified DNA is added to AuNP solution for 5 minutes for initial binding to take place. The addition of a strong salt solution, SSC, will cause the AuNPs to precipitate out of solution if they are able to aggregate. Aggregation is only impeded if the AuNPs are partially bound by DNA, which is resultant of intermediate methylation status. Image credit: Sina et al, doi: 10.1038/s41467-018-07214-w.
The use of gold nanoparticles in cancer biology has been investigated as a therapeutic, diagnostic, and dual theranostic agent.
This aggregation-based method of using AuNPs has previously been used to differentiate single- vs. double-stranded DNA, similarly reliant on their distinct physical properties.
In 2010, a group from Shanghai University developed a gold-based colorimetric method to study the mechanistic process of DNA methylation, however with a focus on microbiology instead of cancer biology.
Cell-free DNA (cfDNA) has been turned to with more frequency as a mechanism for cancer detection, as well as a window through which oncologists can monitor cancers’ progression and resistance over long-term follow-ups.
The minimal invasiveness of the procedure, the liquid biopsy, combined with the capacity for powerful next generation sequencing to identify low-frequency mutant variants that are shed from the cancer and diluted in the circulatory system.
The model described by Sina et al, was capable of successfully recognizing cancer DNA from normal DNA at frequencies as low as 1%. The clinical accuracy is striking as well, reaching 90% using the gold electrode plate model.
This paradigm of looking at epigenome-wide differences in cancer as a question of physics and chemistry is the novel tractable angle that may change the way the field approaches these types of questions.
This model at best is a binary signal indicating the presence of cancerous DNA in a blood sample, and does not provide specific information as to the cancer subtype or other prognostic markers.
Corresponding author Dr. Matt Trau has his eyes to the future, as he is thrilled that this work, “led to the creation of inexpensive and portable detection devices that could eventually be used as a diagnostic tool, possibly with a mobile phone.”
The study was published in the journal Nature Communications on December 4, 2018.
Source: Sci News