Removal of the A10 adenosine in a DNA-stabilized Ag16 nanocluster

The role of the terminal adenosine (A10) on the spectroscopic and structural properties of a previously described DNA-stabilized Ag16 nanocluster (DNA:Ag16NC) is presented. In the original DNA:Ag16NCs (5′-CACCTAGCGA-3′), the A10 nucleobase was involved in an Ag+-mediated interaction with an A10 in a neighboring asymmetric unit, and did not interact with the Ag16NC. Therefore, we synthesized AgNCs embedded in the corresponding 9-base sequence in order to investigate the crystal structure of these new DNA-A10:Ag16NCs and analyze the photophysical properties of the solution and crystalline state. The X-ray crystallography and spectroscopic measurements revealed that the 3′-end adenosine has little importance with respect to the photophysics and structure of the Ag16NCs. Additionally, the new crystallographic data was recorded with higher spatial resolution leading to a more detailed insight in the interactions between the nucleotides and Ag atoms.

Detailed information about the synthesis and HPLC purification of DNA-A10:Ag16NCs.
The HPLC purification was performed using a preparative HPLC system from Agilent Technologies with an Agilent Technologies 1260 infinity fluorescence detector, an Agilent Technologies 1100 Series UV-Vis detector, and a Kinetex C18 column (5 μm, 100 Å, 250 x 4.6 mm). The mobile phase was a gradient mixture of 35 mM triethylammonium acetate (TEAA) in Milli-Q water and methanol. The elution gradient is described in Table S1. The run was followed by 6 min of washing with 95% 35 mM TEAA in methanol to remove any remaining sample from the column. The flow rate was 1.3 mL/min. As shown by the chromatogram in Figure S1, a pure fraction of DNA-A10:Ag16NCs was collected at ~36% 35 mM TEAA in MeOH (around 18 min) by using the absorbance signal at 530 nm.

Quantum yield (Q) determination
The fluorescence quantum yield was determined by a relative method, using a Cresyl Violet in absolute ethanol (QR = 0.56) as reference. 1 The absorption and emission spectra were recorded at different concentrations for the sample and the reference. The integrated emission spectra were then plotted as a function of the fraction of absorbed light at the excitation wavelength (f = 1 -10 -A ). The data was fitted linearly while fixing the y-intercept at zero, and the slopes were used to calculate the quantum yield based on the following equation: where S and R stand for the sample and reference, respectively, Q is the quantum yield, α is the slope of the linear regression and n is the refractive index of the solvent.    The intercept is fixed at zero, and the slope represents the hydrodynamic volume.
TCSPC fits.  Figure 3 and Figures S6 and S7 were acquired with a SONY XPERIA XZ mobile phone camera.
Confocal spectra from individual DNA-A10:Ag16NC crystals were recorded on an inverted confocal microscope (Olympus IX71) equipped with an Olympus CPlanFL N 10x objective. 507.5 nm pulsed laser (LDH-P-C-510, Picoquant) was used as excitation source (10 MHz, 3 nW on top of the sample) in combination with an excitation filter (Semrock FF02-510/10-25) and an emission filter (Semrock BLP01-532R-25). The dichroic filter cube consisted of an Olympus BP510-550 excitation filter, Olympus BA590 emission filter and Semrock FF580-FDi01 dichroic filter. The emission spectra were recorded with a spectrometer (Princeton Instruments SPEC-10:100B/LN_eXcelon CCD camera with SP 2356 polychromator, 300 grooves/mm). X-axis calibration was performed using the emission lines of a neon spectral lamp (6032 Newport), while y-axis calibration was done by measuring a reference spectrum on an intensity-calibrated Fluotime300 (PicoQuant). Emission spectra ( Figure  S8) were acquired with 5 s integration time.