(Georgia Institute of Technology, 2011-05-11)
Tomassi, Taylor Alexandra
In the human body, DNA-linked colloidal assemblies are prone to cleavage by nucleases,
yielding the uncontrolled release of particles and any associated therapeutics. Thus, for in
vivo applications, the DNA-linkages must be protected from cleavage by serum
nucleases. The goal of this research is to stabilize DNA duplexes in the presence of
nucleases by chemically modifying the primary target. The effects of sterically protecting
DNA duplexes from nuclease activity by including a polymeric tail on oligonucleotide
targets and by including LNA bases in the target sequence were investigated. The
variables explored included the effect of tail chemistry as well as tail length on the
kinetics and extent of nuclease activity. In DNA digests, two types of polymeric "tails"
were compared: polyethylene glycol (PEG) chains and single stranded thymine-based
strands (dT). Long and short PEG and thymine tails of equivalent lengths were compared.
Ezymatic digests were also performed on fluorescently labeled primary targets modified
with oligonucleotide analogs called LNA, locked nucleic acids. Flow cytometry was used
to quantify the hybridization activity and measure the probe-target duplex density as well
as to determine time-dependence of nuclease activity by monitoring the number of
duplexes remaining following incubation with DNase I. Significant clipping was
observed for all DNA targets tested and indicated that various polymeric tails did not
significantly hinder nuclease activity. These results indicate that the relatively short
polymeric tail lengths do not have appreciable effects on the hindrance of nuclease
activity. LNA digests, on the other hand, showed enhanced stability of primary duplexes
in the presence of nucleases after 24 hours and suggested that LNA may be used as an
alternative to DNA to stabilize colloidal assemblies for drug delivery.
(Georgia Institute of Technology, 2010-05-11)
Tomassi, Taylor Alexandra
In the human body, DNA-linked colloidal assemblies are prone to cleavage by nucleases, yielding the uncontrolled release of particles and any associated therapeutics. Thus, for in vivo applications, the DNA-linkages must be protected from cleavage by serum nucleases. The goal of this research is to stabilize DNA duplexes in the presence of nucleases by chemically modifying the primary target. The effects of sterically protecting DNA duplexes from nuclease activity by including a polymeric “tail” on oligonucleotide targets and by including LNA bases in the target sequence were investigated. The variables explored included the effect of tail chemistry as well as tail length on the kinetics and extent of nuclease activity. In DNA digests, two types of polymeric "tails" were compared: polyethylene glycol (PEG) chains and single stranded thymine-based strands (dT). Long and short PEG and thymine tails of equivalent lengths were compared. Ezymatic digests were also performed on fluorescently labeled primary targets modified with oligonucleotide analogs called LNA, locked nucleic acids. Flow cytometry was used to quantify the hybridization activity and measure the probe-target duplex density as well as to determine time-dependence of nuclease activity by monitoring the number of duplexes remaining following incubation with DNase I. Significant clipping was observed for all DNA targets tested and indicated that various polymeric tails did not significantly hinder nuclease activity. These results indicate that the relatively short polymeric tail lengths do not have appreciable effects on the hindrance of nuclease activity. LNA digests, on the other hand, showed enhanced stability of primary duplexes in the presence of nucleases after 24 hours and suggested that LNA may be used as an alternative to DNA to stabilize colloidal assemblies for drug delivery.