Microcal origin version 6

Low-temperature (77 K) chlorophyll fluorescence emission spectra of C. priscuii cell cultures and cell fractions were collected as described previously (Szyszka et al., 2007 (link)) using a PTI QM-7/2006 spectrofluorometer (Photon Technology International, South Brunswick, NJ, United States) equipped with double monochromators, R928P red-sensitive photomultiplier tube (Hamamatsu Photonics, Shizuoka-ken, Japan) and a liquid nitrogen cuvette. Cell cultures and cell fractions were frozen in liquid nitrogen in the presence of 30% (v/v) glycerol before the measurements. Corrected fluorescence emission spectra were excited at 436 nm and recorded from 650 nm to 800 nm using slit width of 2.5 nm for both excitation and emission. The fluorescence difference spectra were obtained as described in (Savitch et al., 2011 (link)).
Decomposition analysis of the fluorescence emission spectra in terms of 5 Gaussian bands was carried out by a non-linear least squares algorithm that minimizes the chi-square function using a Microcal Origin Version 6.0 software package (Microcal Software, Northampton, MA, United States). The fitting parameters for the five Gaussian components, that is, position, area, and full width at the half-maximum (FWHM), were free-running parameters.

The data are expressed as mean ± S.E.M. Statistical analysis was performed with the Microcal Origin Version 6 (Microcal Software Inc., Northampton, MA, USA). The data analysis was carried out by one‐way anova analysis, the comparison of mean values of the two groups was performed by Student's t‐test. P values less than 0.05 were considered to be significantly different.

The UV-spectra and thermal denaturation profiles of the PNA/DNA complexes and the DNA duplex were recorded with a Jasco UV 750 spectrophotometer (Japan) and a temperature controlled cell in 5 mM sodium phosphate buffer, 140 mM KCl, 5 mM MgCl₂ (pH 7.46). The concentration of each oligonucleotide or PNA was 5 μM. The samples were denatured at 95°C for 5 min and slowly cooled to 20°C prior to analysis. The absorbance at 260 nm was measured as a function of temperature. The data were recorded every 0.5°C from 5 to 90°C. The melting temperature of the DNA duplex and the mismatched PNA₂DNA triplexes were defined as the maxima of the first derivatives of absorption from temperature using MicroCal (TM) Origin Version 6.0 (Serial Number G73S4-9478-7000000). Circular dichroism (CD) spectra were obtained with a Jasco J-715 spectropolarimeter at 15°C. The samples were annealed in the same buffer and under the same conditions as used in thermal denaturation studies. The CD values (Δε) are provided per mole of nucleotide.

The HPLC data were analysed using MicroCal (TM) Origin Version 6.0 (Serial Number G73S4-9478-7000000).