(T1230-09-51) Direct Monitoring of DNA Plasmid Concentration and the Extent of Plasmid-Liposome Complexation Using 2^nd Derivative Fiber-Optic UV Spectroscopy

Purpose: In this study, plasmid DNA-liposome complexes were characterised by fiber-optic UV-Vis spectroscopy, in a conjunction with 2^nd derivative analysis, with the aim to develop a method for direct monitoring of the extent of plasmid DNA complexation. pcDNA3-clover encoding plasmid was mixed with lipofectamine^TM 2000, a liposomal transfection agent, _­at various concentrations and the UV-Vis spectra obtained were analysed applying 2^nd derivative zero intercept method (ZIM) to allow simultaneous quantification of both the relative concentrations of free and complexed plasmid.

Methods: pcDNA3-Clover plasmid was obtained from addgene.org, amplified for further use as 1 mg/mL plasmid stock solution (from UV at 280 nm). Components for phosphate buffer saline (PBS) buffer (12 mM, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000 was purchased from ThermoFisher (Waltham, MA, USA). The measurements of all UV-Vis spectra were performed using a Rainbow R6 fiber optic system equipped with a Minibath-8 stirring device (Pion Inc.). Spectra processing was performed using AuPRO 7.0 software (Pion Inc.). For plasmid DNA-lipofectamine (P-L) complexation 3 µL 1 mg/mL plasmid in purified nuclease-free water was added to 6 µL of 1 mg/mL lipofectamine 2000 transfection reagent (1:2 mass ratio). After incubation at ambient temperature for 5 minutes, the mixture was diluted in 21 µL of PBS prior to use. Plasmid stock solutions were prepared by 1:10 dilution of 1 mg/mL plasmid stock in PBS. To generate calibration curves, plasmid and P-L complex spectra were obtained from serial addition of required aliquots of stock solutions to 1.5 mL PBS. Plasmid spectra were collected over a concentration range of 0.33 – 1.64 µg/mL, and P-L complex spectra were measured from 0.33 – 1.96 µg/mL. Mixtures of ‘free’ plasmid and P-L complexes were prepared by the addition of plasmid to diluted P-L complexes suspended in PBS buffer at the total DNA concentration of 1.96 µg/mL plasmid. Plasmid concentration was in the first step increased by 0.95 increment, and then by 0.33 µg/mL increments, and spectra recorded at each increment.

Results: Analysis of the plasmid and P-L complex spectra (Figure 1) identified two x-intercept points in the 2^nd derivative spectra, which for plasmid solution occur at 244.11 and 284.37 nm. For the P-L complexes prepared at 1:2 plasmid to lipofectamine mass ratio, the intercepts occur at 248.85 and 287.33 nm, i.e a shift of ~4 nm and ~3 nm relative to plasmid, respectively. The consistency of the 2^nd derivative x-axis intercepts from repeated samples suggests presence of only a single component in the spectrum, but further analysis by an independent method should be conducted to confirmation this. At its x-intercepts of a 2^nd derivative spectrum, an analyte will show no absorbance at any concentration; therefore, a second absorbing analyte in the mixture may be quantitated independently of the first at the wavelength of its own x-intercept. Calibration curves were constructed for the plasmid and for P-L complex from their opposing x-intercept wavelengths (Figure 2). Table 1 shows the resultant concentrations of plasmid and P-L complexes measured in PBS. The initial solution is a mixture of plasmid and P-L complex and additional aliquots of plasmid were added. Concentrations of each component as calculated indicate 44.45% complexation in the initial mixture. Additional aliquots of plasmid do not influence the observed complex concentration.

Conclusion: Analysis of the change in the GelRed UV-Vis spectrum with intercalation to plasmid DNA shows a linear response to the plasmid concentration for both of the studied approaches, but the range can be extended when plotted with the sum of the 2^nd derivative absorption at the intercept points (322 nm, 334 nm). The 2^nd derivative based calibration may be used to calculate the concentration of DNA in a solution containing GelRed, by measuring the change in the shape of the dye spectrum with 2^nd derivative spectroscopy as intercalation occurs, using in situ fiber optics.

References: 1. Son KK, Patel DH, Tkach D, Park A. Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged. Biochim Biophys Acta. 2000 Jun 1;1466(1-2):11-5. doi: 10.1016/s0005-2736(00)00176-0. PMID: 10825426.

Figure 1: 2nd derivative absorption spectra of plasmid (1.64 µg/mL) and plasmid-lipofectamineP-L complex (1.96 µg/mL) in pH 7.4 PBS. Upon complexation with liposomes a ~4 nm shift is observed at each zero x-intercept point of the original plasmid DNA spectrum.



Figure 2: Calibration curves of plasmid and plasmid-lipofectamine complex in PBS. Concentration is plotted against 2nd derivative absorbance at their relative x-axis intercepts (244.11 nm, 248.85 nm) where each exhibit no 2nd derivative response.



Table 1: Concentration of plasmid and P-L complexes in PBS measured by 2nd derivative ZIM method. The initial sample is a mixture of plasmid and P-L complex at total concentration of 1.96 µg/mL. Concentrations of each component calculated by ZIM method are shown in the table (Complexation %). Data clearly indicate that the method allows the distinction of two components, free and complexed DNA.