Spectramax paradigm multi mode detection platform system

Transcription reactions were performed at 37 °C in a total volume of 50 μL. In the absence and presence of 100 μM CK, DNA templates (1.5 μM) were prepared in a buffer containing 40 mM of Tris-HCl (pH 7.6), 8 mM of MgCl₂, 60 mM of KCl, and 3 μM of NMM overnight at 4 °C. Samples were incubated at 90 °C for 5 min and then cooled from 90 °C to 37 °C over 2 h. Then, 0.05% Tween-20, 4 mM of rNTP, 5 mM of dithiothreitol, and 1 U·μL⁻¹ of recombinant RNase inhibitor were added. The fluorescence intensity of NMM at 615 nm was measured at 37 °C using a SpectraMax Paradigm Multi-Mode Detection Platform system (Molecular Devices, CA, USA). After adding 5 U·μL⁻¹ of T7 RNA polymerase, all samples were incubated at 37 °C for 120 min of transcription. DNase I was utilized to digest the DNA templates and after 15 min incubation at 37 °C, the RNA transcripts were mixed with stop solution (80 wt% formamide, 10 mM of Na₂EDTA, and 0.1% blue dextran) for further characterization. Samples were electrophoresed in a 10% polyacrylamide gel containing 7 M of urea in 1×TBE buffer at 120 V for 25 min. As the control, 35, 70, and 90 nucleotides of DNA sequences were electrophoresed. After staining with SYBR Gold (Perkin Elmer Life Sciences, Waltham, MA, USA), scanning was performed with an iBright FL1000 Instrument (Thermo, Woodlands, Singapore).

Fluorescence spectra were performed with a SpectraMax Paradigm Multi-Mode Detection Platform system (Molecular Devices, San Jose, CA, USA) at 37 °C. N-methyl mesoporphyrin IX (NMM) was utilized as the fluorescent ligand with a concentration of 3 μM. Then, 1.5 μM of DNA was dissolved in a buffer containing 40 mM of Tris-HCl (pH 7.6), 8 mM of MgCl₂, and 10, 60, or 100 mM of KCl mixed with 0, 10, 20, 40, 60, 80, 100, 150, or 200 μM of CK at 4 °C. DNA solutions were incubated at 90 °C for 5 min and then cooled from 90 °C to 37 °C over 2 h. The fluorescence spectra of NMM from 550 nm to 750 nm were recorded with the excitation wavelength at 396 nm.
The melting temperature of telo in different molecular crowding conditions was measured with a QuantStudio 5 Real-Time PCR Instrument (Thermo, Woodlands, Singapore). A total of 1.5 μM of telo was dissolved in a buffer containing 40 mM of Tris-HCl (pH 7.6), 8 mM of MgCl₂, 10 mM of KCl and 10 wt% of each cosolute. After incubation at 90 °C for 5 min, all samples were annealed from 90 °C to 4 °C for 2 h. The fluorescence intensity at 623 nm with the excitation wavelength at 470 nm was recorded when samples were heated from 4 °C to 90 °C at a rate of 0.9 °C·min⁻¹.

DNA-AgNCs were synthesized according to a previous reference [28 (link)]. Briefly, 10 μM of DNA was mixed with 60 μM of AgNO₃. After incubating at room temperature for 10 min, 60 μM of fresh NaBH₄ was added with vigorous shaking for 5 min. The solution was kept in the dark at 4 °C for 24 h to form AgNCs. The fluorescence intensity of the samples after 10-fold dilution was measured by a SpectraMax Paradigm Multi-Mode Detection Platform system (Molecular Devices, CA, USA).