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Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors.

ABSTRACT: Here, we describe the characterization of new RNA-cleaving DNAzymes that showed the highest catalytic efficiency at pH?4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The trans-acting DNAzyme variants were also catalytically active. Mutational analysis revealed a conservative character of the DNAzyme catalytic core that underpins the high structural requirements of the cleavage mechanism. A significant advantage of the described DNAzymes is that they are inactive at pH values close to physiological pH and under a wide range of conditions in the presence of monovalent and divalent metal ions. These pH-dependent DNAzymes could be used as molecular cassettes in biotechnology or nanotechnology, in molecular processes that consist of several steps. The results expand the repertoire of DNAzymes that are active under nonphysiological conditions and shed new light on the possible mechanisms of catalysis.

SUBMITTER: Kasprowicz A

PROVIDER: S-EPMC5288747 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors.

Kasprowicz Aleksandra A Stokowa-Sołtys Kamila K Jeżowska-Bojczuk Małgorzata M Wrzesiński Jan J Ciesiołka Jerzy J

ChemistryOpen 20161219 1

Here, we describe the characterization of new RNA-cleaving DNAzymes that showed the highest catalytic efficiency at pH 4.0 to 4.5, and were completely inactive at pH values higher than 5.0. Importantly, these DNAzymes did not require any divalent metal ion cofactors for catalysis. This clearly suggests that protonated nucleic bases are involved in the folding of the DNAzymes into catalytically active structures and/or in the cleavage mechanism. The <i>trans</i>-acting DNAzyme variants were also ...[more]

PMID: 28168150

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Project description:A versatile approach to modify metal-organic framework nanoparticles (NMOFs) with nucleic acid tethers, using the "click chemistry" method is introduced. The nucleic acid-functionalized NMOFs are used to prepare stimuli-responsive carriers of loads (fluorescence probes or anti-cancer drugs). Two different stimuli-responsive nucleic acid-based NMOFs are presented. One system involves the preparation of pH-responsive NMOFs. The NMOFs are loaded with fluorophores or doxorubicin anti-cancer drug and locked in the NMOFs by pH-responsive DNA duplex capping units. At pH = 5.0 the capping units are unlocked, leading to the release of the loads. The AS1411 aptamer is conjugated to the locking units as the targeting unit for the nucleolin biomarker present in cancer cells. The pH-responsive doxorubicin-loaded NMOFs and, in particular, the AS1411 aptamer-modified pH-responsive NMOFs reveal selective, targeted, cytotoxicity toward MDA-MB-231 breast cancer cells. A second system involves the synthesis of NMOFs that are loaded with fluorophores or doxorubicin and capped with metal-ion-dependent DNAzyme/substrate complexes as locking units (metal ion = Mg2+ or Pb2+ ions). In the presence of the respective metal ions, the nucleic acid locking units are cleaved off, resulting in the release of the loads. Also, "smart" Mg2+-ion-dependent DNAzyme capped doxorubicin-loaded NMOFs are synthesized via the integration of the ATP aptamer sequence in the loop domain of the Mg2+-dependent DNAzyme. The unlocking of these NMOFs proceeds effectively only in the presence of ATP and Mg2+ ions, acting as cooperative triggers. As ATP is over-expressed in cancer cells, the "smart" carrier provides sense-and-treat functions. The "smart" ATP/Mg2+-triggered doxorubicin-loaded NMOFs reveal selective cytotoxicity toward MDA-MB-231 cancer cells. Beyond the use of the metal-ion-dependent DNAzymes as ion-responsive locks of drug-loaded NMOF carriers, the DNAzyme-capped fluorophore-loaded NMOFs are successfully applied as functional units for multiplexed ion-sensing and for the design of logic-gate systems.

Dataset Information

Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors.

Publications

Characterization of Highly Efficient RNA-Cleaving DNAzymes that Function at Acidic pH with No Divalent Metal-Ion Cofactors.

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