A sensitive microfluidic platform for a high throughput DNA methylation assay.
DNA methylation is an epigenetic modification essential for normal development and maintenance of somatic biological functions. DNA methylation provides heritable, long-term chromatin regulation and the aberrant methylation pattern is associated with complex diseases including cancer. Discovering novel therapeutic targets demands development of high-throughput, sensitive and inexpensive screening platforms for libraries of chemical or biological matter involved in DNA methylation establishment and maintenance. Here, we present a universal, high-throughput, microfluidic-based fluorometric assay for studying DNA methylation in vitro. The enzymatic activity of bacterial HPAII DNA methyltransferase and its kinetic properties are measured using the assay (K(m)(DNA) = 5.8 nM, K(m)(SAM) = 9.8 nM and Kcat = 0.04 s(-1)). Using the same platform, we then demonstrate a two-step approach for high-throughput in vitro identification and characterization of small molecule inhibitors of methylation. The approach is examined using known non-nucleoside inhibitors, SGI-1027 and RG108, for which we measured IC50 of 4.5 μM and 87.5 nM, respectively. The dual role of the microfluidic-based methylation assay both for the quantitative characterization of enzymatic activity and high-throughput screening of non-nucleoside inhibitors coupled with quantitative characterization of the inhibition potential highlights the advantages of our system for epigenetic studies.
The Mina & Everard Goodman Faculty of Life Sciences, The Nanotechnology Institute, Bar-Ilan University, Ramat Gan, 5290002, Israel. Doron.Gerber@biu.ac.il.
Maria
M
Ronen
Dorit
D
Avrahami
Doron
D
Gerber
D000634:Aminoquinolines / Q000737:chemistry; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D044127:Epigenesis, Genetic; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D010797:Phthalimides / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry
D000634:Aminoquinolines; D001426:Bacterial Proteins; D010797:Phthalimides; D011743:Pyrimidines; C503639:RG108; C581203:SGI-1027; D014364:Tryptophan; D004247:DNA; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases