Conditional Deoxyribozyme Gene Knockdown: Investigating DNA Displacement Reactions on Gold Nanoparticles Open Access

Harrell, Kevin Nicholas (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/b2773w42k?locale=en
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Abstract

Abstract
Conditional Deoxyribozyme Gene Knockdown:
Invesitgating DNA Displacement Reactions on Gold Nanoparticles
Here we present the development of a conditional RNA hydrolysis catalyst with
the potential for cellular gene knockdown through the use of DNA displacement
reactions on gold nanoparticles. The activity of catalytic oligonucleotides, or
deoxyribozymes, that hydrolyze the phosphodiester backbone of specific RNA strands,
is triggered by using a secondary input signal. DNAzymes are widely investigated for
their potential in gene regulation. One of the major challenges pertinent to DNAzymes in
particular, and RNA interference techniques in general, is the ability to act on specific
cell types without affecting others. To overcome the issue of selectivity while also
addressing the need for cellular delivery, we use DNA functionalized gold nanoparticles,
along with DNA displacement reactions to achieve a conditional response which is
triggered by the presence of a specific switch strand. The DNAzyme was shown to be
inactive when hybridized to a capture strand functionalized on a gold nanoparticle, and
active in solution once displaced from the capture particle by the specific switch strand.
The initial rate constant of DNAzyme activity was shown to increase up to 14-fold upon
addition of appropriate concentrations of switch strand. Thus, the catalyst is triggered by
the presence of a switch strand. We have also shown that the system is highly selective
and is unlikely to be activated by other strands. This system has broad potential for
clinical applications in that it can be selectively activated based on a cell's genetic and
molecular profile. Gene knockdown will only occur in the presence of specific mRNA
markers, allowing the system to differentiate between cells. Therapeutic techniques
could be developed from this system to treat or kill cancerous cells while leaving healthy
cells unaltered.

Table of Contents

Table of Contents
Introduction 1


Gene Knockdown 2


Deoxyribozymes 4


DNA Functionalized Gold Nanoparticles 6


Conditional Response Signaling 9


Innovation 10


Experimental 14


DNA Sequences 14


Gold Nanoparticle Synthesis 15


Gold Nanoparticle DNA Functionalization 16


Density Assay 17


Catalytic Experiments 18


Results 20


Solution Studies 20


Nanoparticle Studies 25


Conclusions 32


Future Direction 33


References 34

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