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 137798 samples such as water and soil, with very few demonstrating detection inside cells.
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.
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. Longer exposure to nm light led to greater increase in fluorescent signal. Supplementary Material Supporting 1379 Click here to view.
Since deprotection is performed with light, lej should be orthogonal to cellular delivery and cellular function, and thus allow temporal control over the uncaging and activation of the DNAzyme sensor. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e. Nat Rev Mol Cell Biol.
Depending on the presence of metal cofactors inside and outside of the cells, the DNAzymes may not be able to reach their cellular destination before they are cleaved. An attractive advantage of our photocaging strategy is that we can use the same caged substrate strand to achieve 137798 of different metal ions by using different enzyme strands. DNAzymes, 13978 of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
Author information Copyright and License information Disclaimer. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells. University Science Books; Angew Chem Int Ed. Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells. 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 lek interference is very difficult.
J Biol Inorg Chem. 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.
As a result, despite photolabile group addition having been widely used as a chemical biological tool in the development of photoactivatable proteins, [ 11 ] small molecules, [ 2d11c, 11d12 ] and oligonucleotides, [ 11c, 11d13 ] no such strategy has yet been reported to enable the use of DNAzymes for sensing metal ions in living cells.
As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal. 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.
Figures S5, S6 in SI. Supporting information for this article is given via a link at the end of the document. Metal ions have been involved in le critical functions in biology, providing structural stability and catalytic activity to proteins, and alone as signaling molecules. To overcome this limitation, we demonstrate herein the design and synthesis of a photoactivatable or photocaged DNAzyme, and its application in sensing Lie II in living cells.
In addition to showing the intracellular activation of a DNAzyme metal ion sensor, we also demonstrate that this strategy is applicable towards all members of the broader class of RNA-cleaving DNAzymes, making this work a significant oei towards achieving the use of DNAzymes as a generalizable platform for cellular metal ion detection and imaging.
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.
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This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. Annu Rev Anal Chem. These results strongly suggest that the DNAzyme activity can be restored after light activation: Open in a separate window.
While the addition of photolabile or photoswitchable groups has been used to control the activity of 1378 previously, [ 10 ] no previous report has been able to control both the activity of the DNAzyme and the stability and cleavage of the substrate strand. This feature also allows multiple DNAzymes to recognize lri same substrate sequence. Generalizability of caging strategy. Eur J Inorg Chem.
Curr Opin Chem Biol. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. As a result, the exact substrate sequence llei can be recognized by a DNAzyme can be arbitrarily chosen.
To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. Schlosser K, Li Y. The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. See other articles in PMC that cite the published article.
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.
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Support Center Support Center. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
J Am Chem Soc. Coleman fellowship at the University of Illinois at Urbana-Champaign.