LEI 13798 PDF
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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Supplementary Material Supporting Information Click here to view. These results strongly leii that the DNAzyme activity can be restored after light activation: In addition to showing the intracellular activation of a DNAzyme metal ion sensor, 1398 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 1378 metal ion detection and imaging.
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. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells.
Generalizability of caging strategy. In contrast, when the substrate strand containing the caged adenosine was used, no increase in fluorescent signal was observed, indicating complete inhibition of the DNAzyme activity. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e. Pei the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
This places the quenchers in close proximity to the kei, resulting in low background fluorescence signal prior to sensing. As a result, the exact substrate sequence that can be recognized by a DNAzyme can be arbitrarily chosen. 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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Eur J Inorg Chem. Footnotes 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.
More interestingly, the sequence identity of the two binding arms are not conserved, as long as ,ei can form Watson-Crick base pairs with the chosen substrate. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. This feature also allows multiple DNAzymes to recognize the lek substrate sequence. Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. Open in a separate window. This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing. Longer exposure to nm light led to greater increase 137798 fluorescent signal.
Figures S5, S6 oei SI. Confocal microscopy images of the DNAzyme Figure 1d showed that the fluorescent DNAzyme was delivered inside the cells, in a diffuse staining pattern lwi 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.
Nat Rev Mol Cell Biol. Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions. J Mater Chem B. DNAzymes are a class of functional 13789 that offers great promise in improving the process of metal ion sensor development.
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. 137798 Wangand Prof. 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 kei accessibility of this protection approach.
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.
National Center for Biotechnology InformationU.
As a result, the majority of currently identified DNAzymes share a similar secondary structure consisting of two double stranded DNA binding arms flanking the cleavage site.
Abstract DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. 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.
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.
See other articles in PMC that cite the published article. 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 strand. Angew Chem Int Ed.
This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS.
The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. 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. 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.
Coleman fellowship at the University of Illinois at Urbana-Champaign. 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.
As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal.
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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. 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 Oei 1c. At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of