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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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. The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example.
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. 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.
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, 11d llei, 13 ] no such strategy has yet been reported to enable the use of DNAzymes for sensing metal ions in living cells.
An attractive advantage of our photocaging strategy is that we can use the same caged substrate strand to achieve sensing of different metal ions by using different enzyme strands. Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors.
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This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. 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.
The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells.
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.
Supporting information for this article is given via a link at the end of the document.
Supplementary Material Supporting Information Click here to view. As a result, the exact substrate sequence that can be recognized by a DNAzyme can be arbitrarily chosen. Principles of Bioinorganic Chemistry. 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.
University Science Books; National Center for Biotechnology InformationU. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells.
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Curr Opin Chem Biol. Angew Chem Int Ed. DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development.
These results strongly suggest that the DNAzyme activity can be restored after light activation: Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods. Longer exposure to nm light led to greater increase in fluorescent signal.
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. 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 ldi cells.
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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. This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing. Metal ions have been involved in many 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 Zn II in living 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.