Angew Chem Int Ed Engl. Dec 8; 53(50): – .. Lei Lei, Department of Bioengineering and Institute of Engineering in Medicine, University of. Kevin Hwang, Peiwen Wu, Taejin Kim, Lei Lei, Shiliang Tian, Yingxiao Wang, . Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This work is supported by the US National Institutes of Health (ES to Y.L.) and by the Office of Science (BER), the U.S. Department of.
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Even though the use of DNAzymes for metal ion sensing has been established for some time, the majority of previously published work has been limited to sensing metal ions in environmental samples such as water and soil, with very few demonstrating detection inside cells. Footnotes Supporting information for this article is given via a link at the end of the document. In this way, the DNAzymes can be allowed to enter into cells and distribute in different compartments without being cleaved prematurely.
Recognizing this important connection, we and other labs have taken advantage oei this property to develop corresponding metal ion sensors. A complementary approach to rational design is combinatorial selection, which does not rely on prior knowledge of metal-binding, and in which lfi selectivity and affinity can be improved by adjusting the stringency of selection conditions. Supplementary Material Supporting Information Click here to view.
The performance of the photocaged DNAzyme was first assessed lie a buffer under physiological conditions. Furthermore, the enhanced stability of the caged DNAzyme does not require the use of a specific nanomaterial vehicle as a delivery agent, further demonstrating the wider accessibility of this protection approach.
Confocal microscopy pei of the DNAzyme Figure 1d showed that the fluorescent DNAzyme was delivered inside the cells, in a diffuse staining pattern mainly localized in the nucleus determined by colocalization 137988 Hoechst stain.
While no fluorescent signal increase was observed in the absence of light, the fluorescent signal showed an increase with time after addition of metal ions Figure 1c.
It is thus necessary to develop a method that allows both the controlled activation of the DNAzyme as well as a method for reversibly protecting the RNA cleavage site from enzymatic degradation. This allows the fluorophore to ,ei separated from the quenchers, giving a dramatic increase in fluorescent signal. J Mater Chem B. This work will greatly expand the applicability of DNAzymes as versatile biosensors lie will greatly improve the field of metal ion sensing.
See other articles in PMC that cite the published article.
DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. In contrast, when the substrate strand containing the caged adenosine was used, no increase in fluorescent signal was observed, indicating complete inhibition of the DNAzyme activity.
Principles of Bioinorganic Chemistry.
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Angew Chem Int Ed Engl. Generalizability of caging strategy. National Center for Biotechnology InformationU. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods. This feature also allows multiple DNAzymes to recognize the same substrate sequence. An attractive advantage of our photocaging strategy is that we can use the same caged substrate strand to achieve sensing of different metal ions by using different enzyme strands.
However, most methods rely on rational design, and success in designing one metal sensor may not be readily translated into success for another metal sensor, because the difference between metal ions can be very subtle and designing sensors with high selectivity and little or no interference is very difficult. To overcome this limitation, we demonstrate herein the design and synthesis of a photoactivatable or photocaged DNAzyme, and its llei in sensing Zn II in living cells.
To confirm that the observed increase in 137798 was caused by DNAzyme activity and not nonspecific cleavage by other cellular components, we used an enzyme sequence in which two critical bases in the catalytic loop have been substituted Supplemental Table S1. In conclusion, we have demonstrated a eli and effective strategy for protecting the substrate of a DNAzyme sensor, enabling its delivery into cells without being cleaved during the process, and allowing it to be used as a cellular metal ion sensor upon photoactivation.
Annu Rev Anal Chem. As the only modification to the original DNAzyme is on the substrate strand, we can replace the enzyme strand without needing to re-optimize for each new substrate sequence, greatly improving the generalizability of this protection strategy.
This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing. Longer exposure to nm light led to greater increase in fluorescent signal. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.
Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions. Angew Chem Int Ed.