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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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.
lwi Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance. Schlosser K, Li Y.
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. 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.
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. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells. 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.
Coleman fellowship at the University of Illinois at Urbana-Champaign. 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.
Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
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Depending on the presence of metal cofactors inside and outside of the cells, the DNAzymes may not be lsi to reach their cellular destination before they are cleaved.
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. This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing.
Eur J Inorg Chem.
J Biol Inorg Chem. Angew Chem Int Ed. Annu Rev Anal Chem. Author manuscript; available in PMC Dec 8. The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand.
Longer exposure to nm light led to greater increase in fluorescent signal. Principles of Bioinorganic Chemistry. Supplementary Material Supporting Information Click here to view. Curr Opin Struct Biol. At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing. Author information Copyright and License information Disclaimer. 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.
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. 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. Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions.
University Science Books; This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS.
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. J Am Chem Soc. The selection process allows DNAzymes with specific binding affinity, selectivity, and sensitivity to be obtained. Figures S5, S6 in SI.
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. Nat Rev Mol Cell Biol.
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National Center for Biotechnology InformationU. DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development. Supporting information for this article is given via a link at the end of the document. This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal.
The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. 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.
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To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. Abstract DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. Even llei 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.
Footnotes Supporting information for this article is given via a leo at the end of the document. J Mater Chem B.