Quantification of accessible DNA on vertically aligned carbon nanofibers in cellular delivery systems

Abstract

Vertically aligned carbon nanofibers with tethered plasmids have been developed as a novel tool for the direct physical introduction of nucleic acids (tethered genes) into living cells. Immobilization of DNA to a scaffold, or tethering, can influence the accessibility and transcriptional activity of the DNA template. Therefore, it is necessary to determine the number of accessible gene copies on each nanofiber. In this investigation, polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR) were used as cell-free tools to quantify the number of gene copies tethered to a nanofiber. Using chips of nanofiber arrays with bound template (pd2EYFP-N1 vector), PCR was conducted with a primer set designed to amplify the CMV promoter and eYFP gene region (1603 bp). Using a non-specific binding protocol of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) condensation of DNA base amines to nanofiber carboxylic acids, amplification yields of the promoter/gene region of bound DNA were quantified and compared to controls with no EDC. During the first round of quantification, approximately 3.1times107 gene copies were amplified from each nanofiber chip in the presence of EDC, while 1.6times107 gene copies were amplified from control nanofiber chips in the absence of EDC. In subsequent quantification steps of the same nanofiber chips, DNA yields decreased dramatically (2.4times104 gene copies) on control chips, while chips incubated in the presence of EDC retained DNA (1.1times107 gene copies). We did not expect such a high number of gene copies from the first round of quantification on nanofiber chips in the absence of EDC. The subsequent decrease in gene copies from these samples suggests that perhaps this DNA was non-specifically adsorbed to the nanofibers and can be removed during thermal cycling. Investigations with other tethering strategies are being addressed to evaluate and optimize DNA binding strategies