Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

Abstract

One of the key aspects of surviving cancer is to detect the disease as early as possible, thereby enlarging the window of opportunity of curing it. [4,5] A recent trend for early cancer diagnostics is the pre-symptomatic detection of cancer biomarkers in liquid biopsy samples and, therefore, improving the survival rate. [6,7] Hypermethylated DNA (hmDNA) is one of the typical biomarkers found in liquid biopsy samples of cancer patients. [8,9] The presence of hmDNA in blood and urine is correlated to multiple types of cancer, including gastric, lung and ovarian cancer. [10][11][12][13] From a genetic point of view, DNA methylation takes place predominantly at promotor regions with a relative high amount of cytosine bases that are followed directly by a guanine (CpGs) in the 5′-to-3′ direction, the so-called CpG-rich regions. [13] The methylation of a CpG is an epigenetic alternation in which a methyl group is covalently bonded to the cytosine base at the fifth carbon. By CpG methylation, gene expressions are controlled in cells. As a consequence, methylation can play a role in tumor development when tumor suppressor genes are methylated, thereby repressing the transcription and thus silencing these genes. [14] Hypermethylation refers to the situation that methylation of a promotor region occurs, which in a healthy situation does not occur.Early disease detection as well as disease progress and the effectiveness of a therapy can be monitored by measuring the hmDNA concentration over time. [15] Yet, the concentration of hmDNA, especially in early-stage cancers, can be as low as a few DNA copies per liquid biopsy sample. [16][17][18] The current approach to measure hmDNA employs DNA isolation and bisulfite conversion, making it time-consuming and labor intensive. As a result, the method is not widely applicable in the clinic. Typically, hmDNA is detected in bisulfite-treated DNA samples by quantitative polymerase chain reaction (PCR) (qPCR, of the region of interest; can be cancer dependent). Alternative hmDNA detection approaches that may be suitable for use in point-of-care applications, include electrochemistry, [19] CRISPR [20] and isothermal amplification. [21] The detection of specific hmDNA biomarkers by sequencing or biosensing approaches involves the ability to distinguish between hmDNA and non-methylated DNA. [22][23][24] Commonly a preselection step is applied, and three different approaches exist in order to differentiate between hmDNA and non-methylatedThe preselection of hypermethylated DNA (hmDNA) from liquid biopsy samples is key to enable early-stage cancer diagnostics. Due to limited selectivity of the existing preselection approaches, however, wide integration in the clinic is currently prohibited. Here, it is argued that an affinity method on a surface, such as used in affinity chromatography, can be significantly improved by employing the principles of multivalency and superselectivity. In the here proposed method, a methyl binding domain (MBD) protein immobilized at a surface is used as a recepto...

Determinants of Superselectivity─Practical Concepts for Application in Biology and Medicine

“…That all data sets essentially collapse onto one master curve is indeed predicted by our scaling variable x S (eq ), and while not highlighted in the original study it demonstrates how superselective targeting can be precisely adjusted by tuning one or more parameters of the multivalent system. Recent examples also demonstrate how surfaces with tunable receptor densities can be exploited for the superselective detection of biopolymers, e.g., DNA by its level of methylation as a cancer biomarker. , …”

“…Recent examples also demonstrate how surfaces with tunable receptor densities can be exploited for the superselective detection of biopolymers, e.g., DNA by its level of methylation as a cancer biomarker. 41 , 42 …”