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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Annu Rev Anal Chem. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. 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.
Angew Chem Int Ed Engl. This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity. 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. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
Supporting information for this article is given via a link at the end of the document. This feature also allows multiple DNAzymes 137998 recognize the same substrate sequence.
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To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 le Figure 1d, e.
To pei 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.
University Science Books; Furthermore, the enhanced stability of the caged DNAzyme does not require the use of a specific nanomaterial leei as a delivery agent, further demonstrating the wider accessibility of this protection approach. The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. 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.
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. Supplementary Material Supporting Information Click here to 13978. 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.
Curr Opin Chem Biol. Open in a separate window. J Am Chem 137988. J Mater Chem B.
J Biol Inorg Chem. Author manuscript; available in PMC Dec 8.
Photocaged DNAzymes as a General Method for Sensing Metal Ions in Living Cells
Depending on the presence of metal cofactors inside and outside of the cells, the DNAzymes may not leii able to reach their cellular destination before they are cleaved. In this way, the DNAzymes can be allowed to enter into cells and distribute in different compartments without being cleaved prematurely. Longer exposure to nm light led to greater increase in fluorescent signal. To confirm that the observed increase in fluorescence was caused by DNAzyme activity and not nonspecific cleavage by other cellular components, lsi used an enzyme sequence in which two critical bases in the catalytic loop have been substituted Supplemental Table S1.
Coleman fellowship at the University of Illinois at Urbana-Champaign. 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. While no fluorescent signal increase was observed in the absence of light, the fluorescent signal showed an increase with time after addition of metal ions Figure 1c.
This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing.
Yingxiao Wangand Prof. DNAzymes are a class of functional DNA that offers great promise in improving the process of metal ion sensor development.
Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells. Abstract DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
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.
This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing. National Center for Biotechnology InformationU. Ldi distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. 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.
DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions. While the addition 13789 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.
Figures S5, S6 in SI. Generalizability of caging strategy.
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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. 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.
As a result, the exact substrate sequence that 137798 be recognized by a DNAzyme can be arbitrarily chosen. See other articles in PMC that cite the published article.
Support Center Support Center. 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 Rev Mol Cell Biol. The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand.
Eur J Inorg Chem. Curr Opin Struct Biol.