LEI 13798 PDF

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.

Author: Kazijas Tezilkree
Country: Namibia
Language: English (Spanish)
Genre: Finance
Published (Last): 7 July 2010
Pages: 338
PDF File Size: 11.75 Mb
ePub File Size: 2.41 Mb
ISBN: 358-7-49776-131-8
Downloads: 90824
Price: Free* [*Free Regsitration Required]
Uploader: Douzahn

DNAzymes, sequences of DNA with catalytic activity, have been demonstrated as a potential platform for sensing a wide range of metal ions.

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. Metal ions have been involved in many critical functions in biology, providing structural stability and catalytic activity to proteins, and alone as signaling molecules.

Lfi advances in understanding the role of biological metal ions will require the development of new sensors for many more metal ions. 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 113798 ions can be very subtle and designing sensors with high selectivity and little or no interference is very difficult.

137998

A complementary approach to rational design is combinatorial selection, which does not lri on prior knowledge of metal-binding, and in which sensor selectivity and affinity can be improved by adjusting the stringency of selection conditions. DNAzymes are a class of functional DNA that offers great promise in improving the process of lfi ion sensor development. Recognizing this important connection, we and other labs have taken advantage kei this property to develop corresponding metal ion sensors.

The selection process allows DNAzymes with specific binding affinity, selectivity, and sensitivity to be obtained. 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.

Depending oei 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. Both metal-catalyzed cleavage and nuclease-induced degradation result in loss of dynamic range, negatively affecting the signal-to-background ratio and sensor performance.

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. 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.

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.

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.

  3M 55045 PDF

The sensor design and photocaging strategy is shown in Figure 13789using the 8—17 DNAzyme as an example. 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.

At ambient conditions, the enzyme and substrate strands can hybridize, as the pair has a melting temperature of This places the quenchers in close proximity to the fluorophore, resulting in low background fluorescence signal prior to sensing.

This allows the fluorophore to be separated from the quenchers, giving a dramatic 133798 in fluorescent signal. In this way, the DNAzymes can be allowed to enter into cells and distribute in different compartments without being cleaved prematurely.

lei 13798 pdf to jpg

As with the unmodified DNAzyme, the reactivated uncaged DNAzyme will then cleave the substrate strand leading to a fluorescent signal. Because the DNAzyme is highly specific to the metal ion used, this photoactivation strategy allows detection of metal ions in cells.

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.

The performance of the photocaged DNAzyme was first assessed in a buffer under physiological conditions. The substrate strand containing either caged adenosine or native adenosine was annealed to the enzyme strand. In the absence of nm light, the llei signal increased rapidly only in the case of the unmodified substrate containing the native adenosine Figure 1bsimilar to those observed previously.

In contrast, when the substrate 137988 containing the caged adenosine was used, no increase in fluorescent signal was observed, indicating complete inhibition of the DNAzyme activity. While no fluorescent signal increase eli observed in the absence of light, the fluorescent signal showed an increase with time after addition of metal ions Figure 1c. Longer exposure to nm light led to greater increase in fluorescent signal. These results strongly suggest that the DNAzyme activity can be restored after light activation: Figures S5, S6 in Lwi.

– Lean Enterprise Institute| Lean Production | Lean Manufacturing | LEI | Lean Services |

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. This distribution pattern is in agreement with previous reports demonstrating nuclear accumulation of DNA delivered via cationic liposomes Lipofectamine PLUS.

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. Furthermore, the inactive DNAzyme showed no significant increase in fluorescence over 45 minutes Figure 1d, e.

Together, these results strongly indicate that the caged DNAzyme can be used to detect and image metal ions in living cells. To overcome this limitation, we are currently investigating the design of new ratiometric sensors that may allow for better quantification within cells.

  GARWOOD WHALEY FUNDAMENTAL STUDIES FOR SNARE DRUM PDF

Since the first discovery of DNAzymes in using in vitro selection, many DNAzymes have been obtained using similar selection methods. 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. 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.

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.

This feature also allows multiple DNAzymes to recognize the same substrate sequence. 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. Generalizability of caging strategy. 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.

This strategy provides enhanced stability up to multiple days in serum and allows temporal control over DNAzyme activity. 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.

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. This work will greatly expand the applicability of DNAzymes as versatile biosensors and will greatly improve the field of metal ion sensing. Coleman fellowship at the University of Illinois at Urbana-Champaign.

Supporting information for this article is given via a link at the end of the document. National Center for Biotechnology InformationU.

Angew Chem Int Ed Engl. Author manuscript; available in PMC Dec 8. Yingxiao Wangand Prof. Author information Copyright and License information Disclaimer. See other articles in PMC that cite the published article. Abstract 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. Supplementary Material Supporting Information Click here to view.

Footnotes Supporting information for this article is given via a link at the end of the document. Principles of Bioinorganic Chemistry. University Science Books; J Biol Inorg Chem. Eur J Inorg Chem. Curr Opin Chem Biol. J Am Chem Soc. Curr Opin Lri Biol. Annu Rev Anal Chem. Angew Chem Int Ed. J Mater Chem B. Nat Rev Mol Cell Biol. Schlosser K, Li Y. Support Center Support Center. Please review our privacy policy.