Effect Of Graphene Oxide On Conformation And Function Of Proteins And DNA

Graphene oxide (GO) is a novel nanomaterial whose applications in biologicalsystems have attracted increasing attention and shown great potential, whereas itsbiosafety at the molecular level still lacks the support of adequate experimental studyso far. In this paper, the effects of GO on conformation and function of proteins andDNA were investigated by molecular spectroscopic techniques such as UV–visibleabsorption, fluorescence, circular dichorism (CD) and fourier transform infrared(FT-IR) spectroscopy, combined with in vitro enzymatic activity assay and real-timefluorescent quantitative PCR (RT-qPCR) technology, in order to provide experimentalbasis for the evaluation of its biological safety.With catalase (CAT) as a model protein, by molecular spectroscopic techniquesand in vitro enzymatic activity assay, the effect of GO on conformation and functionof proteins was investigated, respectively. CD coupled with FT-IR and absorptionspectra measurements indicated that GO induced the decrease of α-helical and-turncontent and the increase of-sheet and random coil fraction in CAT, resulting in theloosening and unfolding of the protein skeleton. The internal hydrophobic aminoacids (mainly tryptophan) in the protein were exposed, and their endogenousfluorescence was distinctly quenched, according to synchronous fluorescence andfluorescence emission together with absorption spectra analyses. The spectroscopicresults showed that the tertiary and secondary structure of CAT is changed by GO.The conformational changes of the protein were also found to show a dependence onconcentration and time of GO treatment. Moreover, the activity assay indicated thatCAT activity is inhibited by GO and the inhibition exhibits concentration-andtime-dependence.With calf thymus DNA (ctDNA) as a model DNA, the effect of GO on DNAconformation was investigated by molecular spectroscopic techniques; with the gapAgene from E. coli BW25113genomic DNA as a model DNA, the effect of GO onDNA replication function was investigated by RT-qPCR technology. CD, fluorescenceand absorption spectra measurements all indicated that single intercalation bindingoccurred between GO and ctDNA whose bode may be that the single atomic layered GO sheets intercalated into the planes between the base pairs of the DNA. Theintercalation induced the alteration of secondary structure of ctDNA, weakening theπ–π stacking interaction between its base pairs and loosening its original contractivestructure, but did not lead to the unwinding of its double helical structure.Fluorescence spectra analysis also showed that the exogenous fluorescence of ctDNAwas obviously quenched by GO, and the quenching effect was due to a staticquenching mechanism. In addition, the binding constant between GO and ctDNA wascalculated based on the absorption spectral data. RT-qPCR experiments indicated thatGO caused clear changes in amplification and melting curves of DNA in a RT-qPCR,reducing the PCR amplification efficiency, inhibiting the PCR reaction and resultingin the occurrence of the non-specific amplification reaction. The results showed thatGO causes the change of secondary conformation of DNA, and produces certaininhibition of the PCR reaction (to some extent DNA replication).These studies demonstrated that a certain concentration of GO could causenegative effects on conformation and function of proteins and DNA, and itsapplications in biological systems need to be treated with caution and furtherevaluated.