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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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.
More interestingly, the lek identity of the two binding arms are not conserved, as long as they can form Watson-Crick base pairs with the chosen substrate.
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. This distribution pattern is in 31798 with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine ,ei. Together, these results strongly indicate that the caged Leii can be used to detect and image metal ions in living cells.
J Biol Inorg Chem. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance. As a result, the exact substrate sequence that can be recognized by a DNAzyme can be arbitrarily chosen. Eur J Inorg Chem. Le work will greatly expand 1378 applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing.
At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of J Am Chem Soc. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. National Center for Biotechnology InformationU. 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.
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 kei of this protection strategy.
Schlosser K, Li Y. This feature also allows multiple DNAzymes to recognize the same substrate sequence. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.
Metal ions have been involved in many critical functions in biology, providing structural stability and catalytic activity to proteins, and alone as signaling molecules.
Nat 1398 Mol Cell Biol. Curr Opin Struct Biol. Even though 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.
DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
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 Leu.
Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e. Longer exposure to nm light led to greater increase in fluorescent signal.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
Coleman fellowship at the University of Illinois at Urbana-Champaign. Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions.
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.
University Science Books; The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. J Mater Leu B. Footnotes Supporting information for this article is given via a link at the end of the document.
Angew Chem Int Ed. Curr Opin Chem Biol. As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal. Support Center Support Center.
This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing. 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. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for eli quantification within cells.
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. Abstract DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
These results strongly suggest that the DNAzyme activity can be restored after light ,ei The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand.