Experimental materials and protocols

All spectra were measured using equipment from the Jasco line, and temperature controlled (Jasco Analytical Instruments, Inc., UK, Essex). Absorption spectra were measured using a Jasco V-660 UV-VIS spectrophotometer. Circular and linear dichroism spectra were measured using the Jasco J-815 and J-720 spectrometers. Fluorescence spectra were measured using the FP-6500 fluorometer with excitation wavelengths at 240 and 265 nm. For erythrocyte examination, separation was conducted using a Force 7 Fisher micro-centrifuge, and cell count images were measured in quadruplicate via an Auto M10/EX-DEMO-UNIT cellometer (Peqlab, Germany). Biological reagents used to compile the data library were primarily supplied by Sigma Aldrich, UK (namely the nucleobases, amino acids, haemoglobin and purified calf thymus DNA). Similarly chemical reagents and solvents used (ethanol, phosphate buffered saline) were also supplied by Sigma.UK. M13 DNA was prepared by members of the Biophysical Chemistry group, and fission yeast from the Biology department, (University of Warwick, UK) and used as received. Lastly erythrocyte studies were conducted on 25ml preparations of defibrinated horse blood from TCS Biosciences, UK.

Table 4.1: Standard scanning machine parameters for the measurement of absorbance, LD and CD within the UV-Vis wavelength range.

Min Wavelength / nm 180 Max Wavelength / nm 750 Data Pitch 0.2 Bandwidth ≤ 2.0 Scan Speed / (nm/min) ≤ 200

For each member of the data library (amino acids, nucleotides, DNA and M13 phage), CD and most crucially ultra-violet - visible (UV-Vis) absorbance spectra were taken using purified 18.2 MΩ water as the solvent. All nucleobase samples were taken using 1 mm path-length cuvettes with concentrations as noted in Figure 4.3. All cuvettes used in these studies were constructed from quartz instead of alternative materials such as glass, which interferes with spectral recording. Aromatic amino acid spectroscopy was conducted, for a range of concentrations between 0.01 and 1 mg/ml for each of L- Tryptophan, L-Tyrosine and L-Phenylalanine. With regards to path-length, samples were taken using either a 1cm or 1mm cuvette as specified. Purified haemoglobin samples were treated in a similar manner using 12 μM solutions to establish the absorbance spectrum and 6 μM spectra for the majority of the CD spectrum (180- 360nm, path-length 1mm), the latter recorded primarily to verify protein integrity. However, above 360 nm higher concentrations of 43.3 μM (Mr ~ 16500) were necessary to achieve a significant signal.

During Gaussian spectrum fitting all library absorbance and CD spectral data were converted using the Beer-Lambert Law to ε for UV-Vis absorbance, and Δε for CD, where ε is the wavelength-dependent molar absorptivity coefficient with units of M-1_cm-1_{. This allows us to disregard concentration as a factor since all spectra taken}

from the same sample should be identical.

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒=  𝜀  .𝑝𝑎𝑡ℎ𝑙𝑒𝑛𝑔𝑡ℎ  .𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛  

In general however, CD spectra were collected purely in order to confirm peaks present in the absorbance spectra, and also to potentially uncover additional peaks missed. Collection of spectroscopic data for both calf-thymus DNA and M13 phage required no additional special preparation. The A-DNA conformation was prepared from calf thymus B-DNA stock solution (1 mg/ml, in purified water). Purified water was added first (3.3 ml) to 0.584 ml of this stock DNA solution followed by slow addition of pure ethanol (to a total of 19.4 ml) with careful stirring (to avoid precipitation), to make an 80% ethanol solution at 30 μg/ml DNA. The stock solution was diluted to 30 μg/ml with purified water for the control B-DNA conformation63

.

4.1.1 Cell preparation

Fresh defibrinated equine blood (screened for viral and other pathogens to ensure sterility) and fission yeast samples (stored in simple growth media) were stored at 4 degrees and used as received. Equine RBCs were initially isolated from the blood stock solution by centrifugation at 600g for 10 min at room temperature. Volumes were such that there was a ratio of 1:2 with regards to blood:buffer (e.g. 100 μl of blood required 200 μl of buffer)57

. After the supernatant was removed via careful pipetting, the RBC pellet was resuspended in cold isotonic PBS (0.9% NaCl, 5 mM phosphate buffer, pH 7) by trituration (passing the surrounding solvent over the pellet repeatedly via micropipette, within the preparation vessel) and then re-centrifuged at 600g for another 10 min. The cell pellet was then washed with PBS buffer thrice to completely remove the plasma and buffer layers (washing twice was acceptable if the supernatant was almost clear after the second wash). The final preparation of washed RBCs was resuspended in isotonic buffer at 0.5, 1 and 2% PCV (Packed Cell Volume, volume of blood cells in a given volume of solution). Lysed red blood cell samples were prepared by simply diluting a given concentration of cell pellet into phosphate buffered purified water.

Fission yeast samples were prepared in a similar manner as the intact RBC samples however the yeast cultures were of a much lower cell concentration compared with blood stocks, despite having been grown to the limit possible without inducing stress reactions (grown in 50 ml vessels). Therefore in order to obtain sufficient

quantities of cells to form the final cell pellet, increased quantities of yeast media was required (4ml of yeast media versus <1ml for blood). The resultant cell pellet formed from 4ml of this media was then suspended in 200 μl of purified water to create a stock solution and successive dilutions were taken as specified.