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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The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme 137988. 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.
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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.
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. University Science Books; Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e. 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 lie environmental samples such as water and soil, with very few demonstrating detection inside cells.
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. In the absence of nm light, the fluorescent signal increased rapidly only in the case of the unmodified substrate containing the native adenosine Figure 137798similar to those observed previously.
Figures S5, S6 in SI. Support Center Support Center. Angew Chem Int Ed Engl. Yingxiao Wangand Prof. 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 majority of currently identified DNAzymes share a similar secondary structure consisting of two double stranded DNA binding arms flanking the cleavage site.
While no fluorescent signal increase was observed in the absence of light, the fluorescent signal 133798 an increase with time after addition of metal ions Figure 1c.
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This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing. This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity.
More interestingly, the sequence identity of the two binding arms are not conserved, as 1398 as they can form Watson-Crick base pairs with the chosen substrate. National Center for Biotechnology InformationU. Supporting information for this lek is given via a link at the end of the document.
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.
Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.
Further advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions.
These results strongly suggest that the DNAzyme activity can be restored after light activation: See other articles in PMC that cite the published article. 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 sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example.
The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. This feature also allows multiple DNAzymes to recognize the same substrate sequence.
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This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. Generalizability of caging strategy.
As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a 1379 signal. Metal ions have been involved in many critical functions in biology, providing structural stability and catalytic activity to proteins, and alone as signaling molecules.
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.
Abstract DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. However, most methods rely on rational design, and success in designing one lel 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.
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. Coleman fellowship at the University of Illinois at Urbana-Champaign. While the addition of photolabile or photoswitchable groups has been used to control the activity of DNAzymes previously, lie 10 ] no previous report has been able to control both the activity of the DNAzyme and the stability and cleavage of the substrate strand.
In conclusion, we 113798 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 leo sensor upon photoactivation.
Schlosser K, Li Y. This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. 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.
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. J Mater Chem B. Open in a separate window.