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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These results strongly suggest that the DNAzyme activity can be restored after light activation: Author information Copyright and License information Disclaimer. Angew Chem Int Ed. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. To overcome this major limitation, we present the design and synthesis of a DNAzyme whose activity is controlled by a photolabile group called photocaged DNAzymeand its application for imaging metal ions in cells.
In conclusion, we have demonstrated a general 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. J Mater Chem B.
This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity. University Science Books; 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. In the absence of nm light, the fluorescent signal increased rapidly only in the case of the unmodified substrate containing the native adenosine Figure 1bsimilar to those observed previously.
Figures S5, S6 in SI. 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.
Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.
The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. Footnotes Supporting information for this article 133798 given via a link at the end of the document.
The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. 17398 Rev Mol Cell Biol. Coleman fellowship at the University of Illinois at Urbana-Champaign. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e.
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Yingxiao Wangand Prof. A complementary approach to rational design is combinatorial selection, which does 17398 rely on prior knowledge of metal-binding, and in which sensor selectivity and affinity can be improved by adjusting the stringency of selection conditions.
Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions. Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors.
J Am Chem Soc.
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More interestingly, the sequence lej of the two binding arms are not conserved, as long as they can form Watson-Crick base pairs with the chosen substrate. 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.
Depending on the presence of metal cofactors 133798 and outside of the cells, the DNAzymes may not be able to reach their cellular destination before they are cleaved.
Schlosser K, Li Y. Author manuscript; available in PMC Dec 8. Since deprotection is performed with light, it should be orthogonal to cellular delivery and cellular function, and thus allow temporal control over the uncaging and activation of the DNAzyme leii.
DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development.