Douzilkree Figures S5, in SI. Annu Rev Anal Chem. As a leei, the majority of currently identified DNAzymes share a similar secondary structure consisting of two double stranded DNA binding arms flanking the cleavage site. 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.
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Brajinn While no fluorescent oei increase was observed in the absence of light, the fluorescent signal showed an increase with time after addition of metal ions Figure 1c. 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. Generalizability of caging strategy. Curr Opin Chem Biol.
Already registered? This feature also allows multiple DNAzymes to recognize the same substrate sequence. Eur J Inorg Chem.
Supplementary Material Supporting Information Click here to view. In contrast, when the substrate strand containing the caged adenosine was used, no increase in fluorescent signal was observed, indicating complete inhibition of the DNAzyme activity. This places the quenchers in close proximity to lri fluorophore, resulting in low background fluorescence signal prior to sensing. J Biol Inorg Chem. DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.
This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity. Figures S5, S6 in SI. Coleman fellowship at the University of Illinois at Urbana-Champaign.
The metal ion selectivity of DNAzymes comes from the sequence identity of the loop in the enzyme strand. The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. In conclusion, leii 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.
Nat Rev Mol Cell Biol. Angew Chem Int Ed Engl. 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.
In addition to showing the intracellular activation of a DNAzyme metal ion sensor, we also demonstrate that this strategy is applicable towards all members of the broader class of 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.
Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells. J Am Chem Soc. Supporting information for this article is given llei a link at the end of the document. 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.
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.
This allows the fluorophore to be separated from the quenchers, giving a dramatic increase in fluorescent signal. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.
Principles of Bioinorganic Chemistry. Support Center Support Center. 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. 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.
The sensor design and photocaging strategy is shown in Figure 1ausing the 8—17 DNAzyme as an example. These results strongly suggest that the DNAzyme activity can be restored after light activation: See other articles in PMC that cite the published article. Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods.
As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal.
Yingxiao Wangand Prof. National Center for Biotechnology InformationU. 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.
This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS. 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. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance.
Annu Rev Anal Chem. 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. 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. Open in a separate window.
J Mater Chem B. 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.
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LEI Nº 13.798, DE 09 DE NOVEMBRO DE 2009