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Published on: March 25, 2015
Biomineralization of Human Genomic DNA into ZIF-8, a Zeolite-Like Metal-Organic Framework
I H Shaykhutdinov1, P V Iliasov2, L V Limareva3
1Researcher, Laboratory of New Medical Materials and Technologies, Professional Center for Education and Research in Genetic and Laboratory Technologies; Assistant, Chemistry Department, Pharmacy Institute; Samara State Medical University, 89 Chapaevskaya St., Samara, 443099, Russia.
Abstract:
The aim of the study was to assess the capabilities of human genomic DNA biomineralization into ZIF-8 metal-organic framework (MOF) preserving DNA sequence integrity after the encapsulation cycle and composite dissolving. The study is an initial stage of the project aimed at developing an abiotic vector to be used when working with native nucleic acids of an arbitrary size based on DNA@ZIF-8 composite.
Materials And Methods:
We studied human genomic DNA isolated from lymphocytes of peripheral blood of healthy volunteers using Proba-NK kit (DNA-Technology LLC, Russia). Genomic DNA purity and concentration was estimated spectrophotometrically at 260/280 nm using Tecan Infinity 200 Pro plate reader (Tecan Instruments, Austria). ZIF-8 was synthesized in the physiological conditions (37°C) by mixing zinc salt and 2-methylimidazole aqueous solutions at different molar ratios. Human genomic DNA was encapsulated into ZIF-8 in similar conditions. The obtained MOF and DNA@ZIF-8 composite were studied using X-ray powder diffraction at the Phaser D2 XRPD device (Bruker, USA), and the specific surface area was estimated using Autosorb iQ porosimetry analyzer (Quantachrome, USA). The encapsulated DNA was quantified by dissolving DNA@ZIF-8 composite in the citrate buffer. DNA integrity was assessed by real-time allele-specific PCR (AS-PCR) using the kits for single nucleotide polymorphisms (Lytech, Russia) at the Quantstudio 6 Pro PCR machine (Thermo Scientific, USA). In case of using the kits with electrophoretic detection, the amplification was performed on the thermal cycler T100 (Thermo Scientific, USA).
Results:
The polymer ZIF-8 and DNA@ZIF-8 composite were obtained at different molar ratios of zinc ions and 2-methylimidazole. We characterized their structure and specific surface area. Genomic DNA biomineralization efficacy was found to be about 7-8%. PCR indicated the integrity of non-selectively chosen loci within the biomineralized DNA.
Conclusion:
The study confirmed the possibility of human genomic DNA encapsulation into ZIF-8 metal-organic framework. After the biomineralization, DNA was found to preserve feasibility to be used in studies to investigate genetic constructs.
