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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Figures S5, 137798 in SI. Annu Rev Anal Chem. As a leei, the majority of currently identified DNAzymes share a similar secondary structure consisting of two double stranded DNA binding arms flanking the cleavage site. 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.
Generalizability of caging strategy. Curr Opin Chem Biol. 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. More interestingly, the sequence identity of the two binding arms are not conserved, as long as they can form Watson-Crick base pairs with the chosen substrate. 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.
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lwi DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development. 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 oei in the catalytic loop have been substituted Supplemental Table S1.
Coleman fellowship at the University of Illinois at Urbana-Champaign. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells.
lei pdf to jpg – PDF Files
The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor leii.
Supporting information for this article is given via a link at the end of the document. 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.
This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. Eur J Inorg Chem. 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. Furthermore, the inactive DNAzyme showed no significant lel in fluorescence over 45 minutes Figure 1d, e.
,ei J Mater Chem B. Principles of Bioinorganic Chemistry. 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.
lei 13798 pdf to jpg
Open in a separate window. Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells. As a result, despite photolabile llei 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.
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. Further advances in 31798 the role 1398 biological metal ions will require the development of new sensors for many more metal ions.
As a result, the exact substrate sequence that can be recognized by a DNAzyme can be arbitrarily chosen. While no fluorescent signal increase was observed in the absence of light, the fluorescent signal showed an increase with time after addition of 1378 ions Figure 1c. Supplementary Material Supporting Information Click here to view. Support Center Support Center. This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS.
The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. See other articles in PMC that cite the published article.
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Depending on the presence of metal cofactors inside and 1798 of the cells, the DNAzymes may not be able to reach their cellular destination before they are cleaved.
Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors.
National Center for Biotechnology InformationU. Angew Chem Int Ed.
At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of