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: Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e.
Footnotes Supporting information for this article is given via a link at the end of the document. See other articles in PMC that cite the published article.
lei 13798 pdf to jpg
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.
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.
A complementary approach to rational design is combinatorial selection, which does not rely on prior knowledge of metal-binding, and in which sensor selectivity and affinity can be improved by adjusting the stringency of selection conditions. 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 sensor.
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. DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
The selection process allows DNAzymes with specific binding affinity, selectivity, and lek to be obtained. Open in a separate window. 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.
Coleman fellowship at the University of Illinois at Urbana-Champaign. Supporting information for this article is given via a link at the end of the document. 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.
Curr Opin Struct Biol. National Center for Biotechnology InformationU. At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of Le are a class of functional DNA that offers great promise in improving the process of metal ion sensor development.
lei pdf to jpg – PDF Files
Because the DNAzyme is highly specific to the metal ion 113798, this photoactivation strategy allows detection of metal ions in cells. To overcome this limitation, we demonstrate oei the design and synthesis of a photoactivatable or photocaged DNAzyme, and its application in sensing Zn II in living cells.
University Science Books; Nat Rev Mol Cell Biol. The DNAzyme contains an enzyme strand and a substrate strand, which are all DNA except for a single adenosine ribonucleotide rA in the substrate strand, at the cleavage site. Supplementary Material Supporting Information Click here to view.
Author manuscript; available in PMC Dec 8. The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. J Mater Chem B. Angew Chem Int Ed. Furthermore, the enhanced stability of the caged DNAzyme does not require the use of a specific nanomaterial vehicle as 17398 delivery agent, further demonstrating the wider accessibility of this protection approach. Author information Copyright and License information Disclaimer.
Figures S5, S6 in Lel. An attractive advantage of our leo strategy is that we can use the same caged substrate strand to achieve sensing of different metal ions by using different enzyme strands. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. Angew Chem Int Ed Le. 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.
J Biol Inorg Chem.
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 137998 photoactivation. Yingxiao Wangand Prof. Longer exposure to nm light led to greater increase in fluorescent signal.
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.
In this way, the DNAzymes can be allowed to enter into cells and distribute in different compartments without being cleaved prematurely. Confocal microscopy images 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 with Hoechst stain. To confirm that the observed increase in fluorescence 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.
Principles of Bioinorganic Chemistry.
J Am Chem Soc. Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions 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 step towards achieving the use of DNAzymes as a generalizable platform for cellular metal ion detection and imaging.