Monitoring the multitask mechanism of DNase I activity using graphene nanoassemblies.
Here we have demonstrated that graphene serves as a remarkable platform for monitoring the multitask activity of an enzyme with fluorescence spectroscopy. Our studies showed that four different simultaneous enzymatic tasks of DNase I can be observed and measured in a high throughput fashion using graphene oxide and oligonucleotide nanoassemblies. We have used phosphorothioate modified oligonucleotides to pinpoint the individual and highly specific functions of DNase I with single stranded DNA, RNA, and DNA/DNA and DNA/RNA duplexes. DNase I resulted in fluorescence recovery in the nanoassemblies and enhanced the intensity tremendously in the presence of sequence specific DNA or RNA molecules with different degrees of amplification. Our study enabled us to discover the sources of this remarkable signal enhancement, which has been used for biomedical applications of graphene for sensitive detection of specific oncogenes. The significant difference in the signal amplification observed for the detection of DNA and RNA molecules is a result of the positive and/or reductive signal generating events with the enzyme. In the presence of DNA there are four possible ways that the fluorescence reading is influenced, with two of them resulting in a gain in signal while the other two result in a loss. Since the observed signal is a summation of all the events together, the absence of the two fluorescence reduction events with RNA gives a greater degree of fluorescence signal enhancement when compared to target DNA molecules. Overall, our study demonstrates that graphene has powerful features for determining the enzymatic functions of a protein and reveals some of the unknowns observed in the graphene and oligonucleotide assemblies with DNase I.
Neil M
NM
Robertson
Mustafa Salih
MS
Hizir
Mustafa
M
Balcioglu
Muhit
M
Rana
§Department of Science, BMCC, City University of New York, 199 Chambers Street, New York, New York 10007, United States.
Hasan
H
Yumak
§Department of Science, BMCC, City University of New York, 199 Chambers Street, New York, New York 10007, United States.
Ozgur
O
Ecevit
Mehmet V
MV
Yigit
D015374:Biosensing Techniques; D004277:DNA, Single-Stranded / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000032:analysis* / Q000737:chemistry; D006108:Graphite / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D010087:Oxides; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D010087:Oxides; D054735:Phosphorothioate Oligonucleotides; D011994:Recombinant Proteins; D012313:RNA; D006108:Graphite; D003850:Deoxyribonuclease I