Investigating steric protection of DNA in the presence of nucleasesReportar como inadecuado

Investigating steric protection of DNA in the presence of nucleases

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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 invivo applications, the DNA-linkages must be protected from cleavage by serumnucleases. The goal of this research is to stabilize DNA duplexes in the presence ofnucleases by chemically modifying the primary target. The effects of sterically protectingDNA duplexes from nuclease activity by including a polymeric tail on oligonucleotidetargets and by including LNA bases in the target sequence were investigated. Thevariables explored included the effect of tail chemistry as well as tail length on thekinetics and extent of nuclease activity. In DNA digests, two types of polymeric -tails-were compared: polyethylene glycol PEG chains and single stranded thymine-basedstrands dT. Long and short PEG and thymine tails of equivalent lengths were compared.Ezymatic digests were also performed on fluorescently labeled primary targets modifiedwith oligonucleotide analogs called LNA, locked nucleic acids. Flow cytometry was usedto quantify the hybridization activity and measure the probe-target duplex density as wellas to determine time-dependence of nuclease activity by monitoring the number ofduplexes remaining following incubation with DNase I. Significant clipping wasobserved for all DNA targets tested and indicated that various polymeric tails did notsignificantly hinder nuclease activity. These results indicate that the relatively shortpolymeric tail lengths do not have appreciable effects on the hindrance of nucleaseactivity. LNA digests, on the other hand, showed enhanced stability of primary duplexesin the presence of nucleases after 24 hours and suggested that LNA may be used as analternative to DNA to stabilize colloidal assemblies for drug delivery.

Undergraduate Research Option Theses - School of Materials Science and Engineering Undergraduate Research Option Theses -

Autor: Tomassi, Taylor Alexandra - -


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