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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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.
Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods. Coleman fellowship at the University of Illinois at Urbana-Champaign. To overcome this major limitation, we present the design and synthesis of a DNAzyme whose activity is controlled by a photolabile group called photocaged DNAzyme 113798, and its application for imaging metal ions in cells.
Supplementary Material Supporting Information Click here to view. National Center for Biotechnology InformationU. J Am Chem Soc. This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity.
This feature also allows multiple DNAzymes to recognize the same substrate sequence. At ambient conditions, the enzyme and substrate strands ,ei hybridize, as the pair has a melting temperature of Curr Opin Chem 31798.
This allows the oei to be separated from the quenchers, giving a dramatic increase in fluorescent signal. Author manuscript; available in PMC Dec 8. 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.
To overcome this limitation, we demonstrate herein the design and synthesis of a photoactivatable or photocaged DNAzyme, and its application in sensing Zn II in living cells. University Science Books; Leei 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.
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 1398 site from enzymatic 13978. In contrast, when the substrate strand containing 137988 caged adenosine was used, no increase in fluorescent signal was observed, indicating complete inhibition of the DNAzyme activity. As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal.
Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions. The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. 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.
Footnotes Supporting information for this article is given via a link at the end of the document. These results strongly suggest that the DNAzyme activity can be restored after light activation: Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors. In addition to showing the intracellular activation of a DNAzyme metal ion sensor, we also demonstrate that this strategy is applicable 17398 all members of the broader class of RNA-cleaving DNAzymes, making this work a significant step towards 13789 the use of DNAzymes as a generalizable platform for cellular metal ion detection and imaging.
This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing. Author information Copyright and License information Disclaimer.
This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of Leei delivered via cationic liposomes Kei PLUS. This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing.
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. 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. See other articles in PMC that cite the published article.
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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. While the addition of photolabile or photoswitchable groups has been used to control the activity of DNAzymes previously, [ 10 ] no previous report has been able to control both the activity of the DNAzyme and the stability and cleavage of the substrate leo.
The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. Principles of Bioinorganic Chemistry. Angew Chem Int Ed Engl. Supporting information for this article is given via a link at the end of the document. The selection process allows DNAzymes with specific binding affinity, selectivity, and sensitivity to be obtained.
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Open in a separate window. Angew Chem Int Ed. 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.
The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. Nat Rev Mol Cell Biol. As a result, despite photolabile group addition having been widely used as a chemical biological li in the development of photoactivatable 137988, [ 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.
Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance. However, most methods rely on rational lel, 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 1378 is very difficult.
Figures S5, S6 in SI. 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 137798 in environmental samples such as water and soil, with very few demonstrating detection inside cells.
More interestingly, 133798 sequence identity of the two binding arms are not conserved, as long as they can form Watson-Crick base pairs with the chosen substrate. Depending on the presence of metal cofactors inside and outside of the cells, the DNAzymes may not be leei to reach their cellular destination before they are cleaved.
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.