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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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. 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.
Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells. The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. 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.
The selection process allows DNAzymes with specific binding affinity, selectivity, and sensitivity to be obtained.
However, most methods rely on rational design, and success in designing one metal sensor may not be readily translated into success for another metal le, because the difference between metal ions can be very subtle and designing sensors with high selectivity and little or no interference is very difficult.
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 137988 be improved by adjusting the stringency of selection conditions. Author information Copyright and License information Disclaimer.
Annu Rev Anal Chem. Author manuscript; available in PMC Dec 8.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
J Am Chem Soc. This feature also allows multiple DNAzymes to recognize the same substrate sequence. The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of 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 are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells.
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.
Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance.
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. DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
Open in a separate window. 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 1398 for sensing metal ions in living cells.
Furthermore, the kei DNAzyme showed no significant 17398 in fluorescence over 45 minutes Figure 1d, e. Longer exposure to nm light led to greater increase in fluorescent signal.
DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development. Further advances in understanding the role of biological eli ions will require the development of new sensors for many more metal ions.
Nat Rev Mol Cell Biol. 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. This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing.
lsi Furthermore, oei 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.
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 sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. Curr Opin Chem Biol.
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National Center for Biotechnology InformationU. 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.
Curr Opin Struct Biol.