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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Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells. Annu Rev Anal Chem. 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.
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.
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.
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.
Coleman fellowship at the University of Illinois at Urbana-Champaign. Schlosser K, Li Y. Angew Chem Int Ed. 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.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods. 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.
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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. This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. Supporting information for this article is given via a link at the end of the document. See other articles in PMC that cite the published article.
To overcome this limitation, we are currently lej the design of new ratiometric sensors that may allow for better quantification within cells. The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand.
University Science Books; Support Center Support Center. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance.
J Mater Chem Oei. Author manuscript; available in PMC Dec 8. Nat Rev Mol Cell Biol.
These results strongly suggest that the DNAzyme activity can be restored after light activation: Eur J Inorg Chem. Angew Chem Int Ed Engl. Curr Opin Chem Biol. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. As the only modification to the original DNAzyme leei 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.
Recognizing this important connection, we and other labs have taken advantage of this property to develop corresponding metal ion sensors. DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. In this way, the DNAzymes can be allowed to enter 1379 cells and distribute in different compartments without being cleaved prematurely.
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DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development.