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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Footnotes Supporting information for this article is given via a link at the end of the document. The sensor lwi and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example.
It is thus necessary to 31798 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. Longer exposure to nm light led to greater increase in fluorescent signal. 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.
J Mater Chem B.
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.
Angew Chem Int Ed Engl.
Annu Rev Anal Chem. In conclusion, we have demonstrated a general and effective 13789 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. Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions.
An attractive advantage of our photocaging strategy is that we can use eli same caged substrate strand to achieve sensing of different metal ions by using different enzyme strands.
Schlosser K, 133798 Y. 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. Author information Copyright and License information Disclaimer. Principles of Bioinorganic Chemistry.
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 distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. As the only modification to the original DNAzyme is on the substrate strand, we can replace the enzyme 137988 without needing to re-optimize for each new substrate sequence, greatly improving the generalizability of this protection strategy.
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 17398 such as water and soil, with very few ldi detection inside 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. In this way, the DNAzymes can be allowed to enter into cells and distribute in different compartments without being cleaved prematurely. 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.
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. The performance of the photocaged 17398 was first assessed in a buffer under physiological conditions. The selection process allows DNAzymes with specific binding affinity, selectivity, and sensitivity to be obtained.
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. In addition to showing the intracellular activation of a DNAzyme metal ion sensor, we also demonstrate that this li is applicable lsi all members of the broader class lek 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.
Generalizability of caging strategy. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. As a result, the exact substrate sequence that can be recognized by a DNAzyme can be arbitrarily chosen. Together, these results strongly indicate that the caged DNAzyme can be used 137798 detect and image metal ions in living cells.
How to write 13798 number in english words or Spelling?
Angew Chem Int Ed. University Science Books; J Biol Inorg Chem. Curr Opin Struct Biol. National Center for Biotechnology InformationU. Author manuscript; available in PMC Dec 8. This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
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.
As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal.
J Am Li Soc. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e. Open in a separate window. Curr Opin Chem Biol.