2600 spectrophotometer

Polyacrylamide gel electrophoresis (PAGE)-purified grade DNA oligonucleotides were purchased from Helix Biosciences. The high-performance liquid chromatography (HPLC)-purified peptide VAQKW was synthesized, and its electrospray ionization-mass spectrometry (ESI-MS) spectra and HPLC chromatogram are given in Fig. S1 and S2,† respectively. The concentration of the peptide was determined by measuring the absorbance of Trp at the C-terminal at 280 nm and 25 °C. The concentrations of single-strand DNA oligonucleotides were determined by measuring the absorbance at 260 nm at a high temperature using a Shimadzu 2600 spectrophotometer (Shimadzu, Tokyo, Japan) connected to a thermoprogrammer. Single-strand extinction coefficients were calculated from mononucleotide and dinucleotide data using the nearest neighbour approximation.^{24,25 (link)}

The amber absorption study was carried out using a Shimadzu-2600 spectrophotometer (Shimadzu, Kyoto, Japan) designed to obtain absorption, transmission, reflection, and energy spectra. The excitation was carried out in the wavelength range of 200–800 nm. Polished Baltic amber was used to study the absorption of amber. A total of 1081 g of amber was weighed on an ALC-210d4 (Aculab, Norwood, MA, USA) laboratory analytical scale, which was then placed in a 10 × 10 mm cuvette for spectrophotometry with transparent walls, and then, together with the sample, the cuvette was placed in the sample holder of the instrument. The sample spectrum was taken using the UVProbe program in “Spectrum” mode. After that, the obtained absorption spectrum of the sample was recorded in the txt format, which was further processed in the Origin program.

The fully reduced SmTGR spectrum has proven difficult to obtain. Low reduction potential, non-native reductant molecules do not readily reduce SmTGR (data not shown). In addition, the enzyme did not tolerate photoreduction in the presence of EDTA (Massey et al., 1978 (link); Massey and Hemmerich, 1978 (link)). Furthermore, when utilizing NADPH as the source of electrons the fully reduced state of the enzyme is not achieved. The pH titration demonstrates that when using the H571A variant the formation of the thiolate-FAD charge transfer can be diminished at more acidic pHs. Therefore, the SmTGR H571A variant was utilized to obtain the spectrum of fully reduced SmTGR. In an anaerobic cuvette, a spectrum of ∼11.5 µM H571A diluted into 1X MAT buffer, pH 5.0, was collected using a Shimadzu 2600 spectrophotometer. The enzyme was mixed with 1 mM NADPH (∼65.8 µL added, prepared in tris base). Repeated spectra were collected until the solution was virtually unchanging. Once the enzyme was fully reduced, the solution was then mixed with 6X MAT buffer, pH 10.28 (∼1 mL) to demonstrate the formation of the broad charge transfer absorption in more basic conditions. After this spectrum was collected, the anaerobic cuvette was opened, and the pH of the final solution was measured to be ∼9. All spectra were corrected for dilution.

A field emission scanning electron microscope (FE-SEM, ZEISS Sigma 500VP) was used for checking the surface morphologies of the different samples. Elemental analysis was performed by energy-dispersive X-ray spectroscopy (EDS) analysis using equipment fixed on the SEM and operated with an acceleration voltage of 15 kV. High-resolution transmission electron microscope (HRTEM) images were taken by JEOL JEM-2100 electron microscope, where sample was prepared for analysis by scratching from titanium substrate, then ultrasonicated with ethanol. X-ray diffraction (XRD) was performed using a (Bruker D8 Advance, Germany) X-ray powder diffractometer, using Cu Kα radiation (wavelength 1.54 A, 40 mA, 45 kV). The Shimadzu 2600 spectrophotometer, Japan with Ba₂SO₄ as the reflectance standard was used for UV-vis analysis. Photoluminescence (PL) spectra were measured using JASCO (FP-8300) spectrofluorometer.

UV-visible absorption spectra were measured on a Shimadzu 2600 spectrophotometer with an ISR-2600Plus Integrating Sphere Attachment, so that the transmittance (T) and the reflectance (R) of the films could be measured. The absorbance of the samples was calculated from −log10 T, while the absorptance was calculated from 1-T-R. Fluorescence spectra were measured with a Horiba Jobin Yvon Fluorolog-2 fluorometer.

The UV absorbance of different samples was recorded with a Shimadzu 2600 spectrophotometer (Shimadzu, Tokyo, Japan) equipped with a temperature controller. Melting curves of DNA structures were obtained by measuring the UV absorbance at 260 nm or 295 nm in 30 mM sodium cacodylate buffer pH (7.0) containing 0.5 mM EDTA, 100 mM NaCl, 100 mM KCl, with and without 40 wt% PEG 200 in the presence or absence of BLM HRDC peptide at different DNA : peptide ratios. The T_m values for 4 μM DNA structures were obtained from the UV melting curves as previously described.^{24,25 (link)} The heating rate was at 0.5 °C min⁻¹. Before measurement, the samples were heated to 95 °C in a water bath, slowly cooled to room temperature, and then incubated at 4 °C overnight to prevent the formation of any structures under non-equilibrium conditions. Experiments were repeated in triplicate to reproduce the data.