Inability of DNAzymes to cleave RNA in vivo is due to limited Mg $$^{2+}$$ 2 + concentration in cells

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg $$^{2+}$$ 2 +... Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10–23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg $$^{2+}$$ 2 + bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg $$^{2+}$$ 2 + ions more strongly and already stoichiometric amounts of Mg $$^{2+}$$ 2 + and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg $$^{2+}$$ 2 + concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg $$^{2+}$$ 2 + concentrations than that available inside the cell. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Biophysics Journal Springer Journals

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg $$^{2+}$$ 2 + concentration in cells

Loading next page...
 
/lp/springer_journal/inability-of-dnazymes-to-cleave-rna-in-vivo-is-due-to-limited-mg-2-2-u0DggU5pUP
Publisher
Springer International Publishing
Copyright
Copyright © 2017 by European Biophysical Societies' Association
Subject
Life Sciences; Biochemistry, general; Biological and Medical Physics, Biophysics; Cell Biology; Neurobiology; Membrane Biology; Nanotechnology
ISSN
0175-7571
eISSN
1432-1017
D.O.I.
10.1007/s00249-017-1270-2
Publisher site
See Article on Publisher Site

Abstract

Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10–23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg $$^{2+}$$ 2 + bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg $$^{2+}$$ 2 + ions more strongly and already stoichiometric amounts of Mg $$^{2+}$$ 2 + and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg $$^{2+}$$ 2 + concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg $$^{2+}$$ 2 + concentrations than that available inside the cell.

Journal

European Biophysics JournalSpringer Journals

Published: Dec 16, 2017

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off