The effect of inosine on the spectroscopic properties and crystal structure of a NIR-emitting DNA-stabilized silver nanocluster

The effect of replacing guanosines with inosines in the two stabilizing strands (5′-CACCTAGCGA-3′) of the NIR emissive DNA-Ag16NC was investigated. The spectroscopic behavior of the inosine mutants is position-dependent: when the guanosine in position 7 was exchanged, the nanosecond fluorescence decay time shortened, while having the inosine in position 9 made the decay time longer. Thanks to structural information gained from single crystal X-ray diffraction measurements, it was possible to propose a mechanistic origin for the observed changes.


Absorption measurements
Absorption spectra were measured with a Cary 300 UV-Vis spectrophotometer from Agilent Technologies using a deuterium lamp for ultraviolet radiation and a tungsten-halogen lamp for visible and near-infrared (NIR) radiation.

Quantum yield measurements and calculations
Quantum yield (Q) values for the inosine mutants were determined at 25 ⁰C in both 10 mM NH4OAc H2O and D2O solutions, using Cresyl Violet in absolute ethanol as a reference (Q=0.56). 2 Absorption and emission spectra of the inosine mutants and the reference compound were measured at different concentrations in order to calculate the corresponding Q, according to the following formula 2 : where Q represents the quantum yield, F is the integrated emission spectrum (i.e., the area under the fluorescence spectrum), fA defines the fraction of absorbed light at the excitation wavelength (532 nm), and n is the refractive index of the medium where the compounds are dissolved in during the measurements. Changes in the refractive index due to isotope variations are ignored since they are in the 1% difference range. The subscripts NC and ref indicate the DNA-AgNC and the reference dye, respectively.

3.2.3
Time-correlated single photon counting (TCSPC) measurements and data analysis Time-resolved fluorescence and anisotropy measurements were performed exciting with a vertically-polarized (V) 507.5 nm pulsed laser at three different temperatures: 5, 25, and 40 ⁰C. Fluorescence decay curves were measured every 5 nm in the 630 -830 nm range. The integration time was varied between 15 and 40 s, whereas the laser repetition rate was selected from 10.0 to 20.0 MHz in order to reach at least 10,000 counts in the maximum at . 3 The analysis of time-resolved data was performed with FluoFit v.4.6 software from PicoQuant. All decay curves were globally fitted with a mono-or multi-exponential reconvolution model including the instrument response function (IRF). The fluorescence intensities at three discrete times after the excitation pulse (0.1, 1, 10 ns) were plotted as a function of emission wavelength in order to construct time-resolved emission spectra (TRES). TRES were then corrected for the detector efficiency and interpolated with a spline function using the built-in spaps MATLAB function with a tolerance of 10 -10 (i.e., forcing the interpolated curve to go through the data points). Time-resolved anisotropy measurements were carried out by recording parallel (VV) and perpendicular (VH) fluorescence decays at λem=730 nm. In order to reach at least 10,000 counts in the maximum, the integration time was varied between 15 and 60 s, while the laser repetition rate ranged from 10.7 to 20.0 MHz. The decay curves were fitted with FluoFit v.4.6 from PicoQuant using, respectively, a multi-exponential and a mono-exponential reconvolution model for the decay time and the rotational correlation time (θ), including the IRF. Based on the Perrin equation 3 , θ values were plotted as a function of η/kBT, where η is the dynamic viscosity of the solvent, kB is the Boltzmann constant and T is the absolute temperature. The data was fitted linearly while fixing the y-intercept at zero. The resulting slope was the hydrodynamic volume (Vhydro) of the clusters (assumed to be spherical).

3.2.4
Steady-state and fluorescence decay measurements at -196 ⁰C Low temperature steady-state and time-resolved emission measurements were carried out only in a 10 mM NH4OAc D2O solution by immersing a NMR tube with the sample in a transparent Dewar filled with liquid nitrogen (-196 °C). The Dewar was then placed in the cuvette holder of a FluoTime300 instrument from PicoQuant. In order to limit the large scattering, due to the presence of ice, two filters were used: a 510 nm band-pass filter (Semrock, FF02-510/10-25) in the excitation path and a 532 nm long-pass (Semrock, BLP01-532R-25) in the emission path. Fluorescence intensity decays, as well as steady-state emission spectra, were measured exciting with a 507.5 nm pulsed laser. Emission spectra were then corrected for the detector efficiency. Fluorescence decay curves were measured every 5 nm in the 610 -850 nm range. The integration time was set to 10 s, whereas the laser repetition rate was selected between 13.3 and 20.0 MHz in order to reach at least 10,000 counts in the maximum at . The data analysis was carried out with FluoFit v.4.6 software (PicoQuant). The decays were globally fitted with a tri-exponential reconvolution model including the IRF. The amplitude (αi) and decay time (τi) components were used to calculate the intensity-averaged decay time, <τ>, 3 and the corresponding intensity as a function of emission wavelength. <τw>, i.e., the overall intensity-weighted average decay time, was then calculated as the average of <τ> over the emission spectra weighted by the steady-state intensity. Furthermore, the background amplitude of the decays (Bkgr) was plotted as a proxy for the microsecond-lived emission. Both the background amplitude and the intensity of <τ> were corrected for the detector efficiency. Finally, the steady state intensity (I) was calculated by using the following formula: 3.2.5 Microsecond decay measurements Microsecond decays were acquired for all inosine mutants in a 10 mM NH4OAc D2O solution at 25 and -196 ⁰C. Details on the low temperature measurements are reported in the previous paragraph. The samples were excited at 508 nm with a Xenon flash lamp (repetition rate = 300 Hz), and the decay curves were recorded at 820 nm with an integration time of 10 min. The decays at 25 ⁰C ( Figure S10) and -196 ⁰C ( Figure S12) were tail-fitted with a mono-and biexponential function, respectively, with FluoFit v.4.6 software from PicoQuant. The microsecond decay times, <τµs>, can be found in Table 2.

Home-built microscope setup
Nanosecond fluorescence and long-lived luminescence of inosine-modified DNA-Ag16NCs were simultaneously measured on our home-built confocal microscope in a burst mode approach described in detail previously. 4 In brief, the method relies on exciting the sample with micro-to millisecond bursts of a high repetition rate excitation light source, which, within its on-time, builds up a population of long-lived luminescent states that decays without interfering with fluorescence, once the excitation source is turned off. At the same time, this approach allows to determine the nanosecond-lived fluorescence. This cycle is repeated until sufficient photons are collected to construct the nano-and micro-second decay curves. By using diverse gating schemes, it is possible to extract pure fluorescent and long-lived luminescent photons. 4 Co-illumination measurements were additionally performed to investigate the optically activated delayed fluorescence (OADF) contribution of the DNA-AgNCs. A description of the optical setups used for single and co-illumination measurements will follow.

Single wavelength excitation setup
For single wavelength measurements, a fiber coupled (FD7-PM, NKT Photonics) pulsed 11 MHz continuum white-light laser (SuperK EXTREME EXB-6, NKT Photonics) was used as an excitation source delivering a wavelength of 520 nm by sending the continuum output through an acoustooptic tunable filter (AOTF; SuperK SELECT, NKT Photonics). The output of the fiber was expanded and collimated by a lens system and cleaned up by a 520 nm band-pass filter (FF01-520/5-25, Semrock) and a 561 nm short-pass filter (SP01-561RU-25, Semrock). Then it was reflected by a 30:70 beam splitter (XF122, Omega Optical) and sent through an air objective (CPlanFLN 10x, NA = 0.3, Olympus), which focused the laser onto the sample and collected the luminescence. The laser light was blocked by a 561 nm long-pass filter (BLP01-561R-25, Semrock) and out-of-focus light was blocked by a 100 µm pinhole. To increase the long-lived luminescence contribution relative to the fluorescence, an 850 nm band-pass filter (FF01-850/10-25, Semrock) was inserted in the emission path. The resulting emission was detected on an avalanche photodiode (CD3226, PerkinElmer) connected to a single photon counting module (SPC-830, Becker & Hickl).

Co-Illumination Setup
For co-illumination measurements, a second excitation path of 850 nm was introduced. The AOTF has two crystals, one for producing visible wavelengths and another for near infrared (NIR) wavelengths. Thus, the 850 nm output from the NIR port of the AOTF was cleaned with an 850 nm band-pass filter (FF01-850/10-25, Semrock) and directed towards the primary excitation beam. The two beams (520 nm and 850 nm) were combined with a dichroic mirror (TLP01-501-25x36, Semrock) and subsequently reflected by a second dichroic mirror (TLP01-628-25x36, Semrock) and sent through an oil immersion objective (UPlanSApo 100x, NA = 1.4, Olympus) that focused the laser onto the sample and collected the luminescence. Compared to the single wavelength excitation setup, a 750 nm short-pass filter was used in the emission path, replacing the 850 nm band-pass filter (FF01-850/10-25, Semrock). The 520 nm beam went through an optical fiber. Due to the optical path length differences between the two beams, the secondary pulse appears 45 ns delayed with respect to the primary.

X-Ray data collection
Single crystal X-ray diffraction measurements were carried out at 100 K with the synchrotron radiation at the BL-17A beamline in the Photon Factory (Tsukuba, Japan) facility. An X-ray beam with a wavelength of 0.90 or 0.98 Å was used for the data collection. The data sets were recorded using 1⁰ oscillation with 0.1 s exposure per frame. This collection time was found to be optimal, since no significant radiation damage was observed for the crystals.

Structure determination and refinement
The data sets were processed by the program XDS. 5 The initial phases were determined with AutoMR from the Phenix suite 6-8 by molecular replacement using the original DNA-Ag16NC structure as a model (PDB-ID = 6JR4). Molecular models were then constructed by using the program Coot. 9, 10 The atomic parameters were refined by the program phenix.refine of the Phenix suite 11 at maximum resolutions of 1.1 and 1.9 Å, for I7_DNA-Ag16NC and the I7-I9 mutant, respectively. Statistics of data collection and structure refinement are summarized in Table S1.

Spectroscopic characterization
Bright-field and fluorescence images were recorded on an inverted Olympus IX71 microscope. For the I7 mutant crystals, a 10x objective (CPlanFL N 10x, NA = 0.3, Olympus) was used, while a 20x objective (LCAch N 20x, NA = 0.40, Olympus) was employed for I7-I9_DNA-Ag16NC. For the bright-field images, white light was used. For the fluorescence images, an X-Cite Series 120Q light source was utilized in combination with an Olympus BP510-550 excitation filter, Olympus BA590 emission filter and Semrock FF580-FDi01 dichroic filter. All images (Figures S2 and S6) were recorded with the camera of an iPhone SE.
Fluorescence decay time measurements of individual crystals (Figures S4 and S8) were carried out on our home-built setup identical to that described in section 3.3.1 with the exception of the objective, which was exchanged to a 40x objective (LUCPlanFL N 40x, NA = 0.60, Olympus). All fluorescence decays were tail-fitted with a bi-exponential model with Origin 2020 software. Steady-state emission spectra of individual crystals (Figures S3 and S7) were recorded on the same setup, but the emission was instead redirected through a spectrograph (SP 2356 spectrometer, 300 grooves/mm, Acton Research) onto a nitrogen cooled CCD camera (SPEC-10:100B/LN-eXcelon, Princeton Instruments). The emission spectra were wavelength-and intensity-calibrated as previously described. 12 HPLC Chromatograms Figure S1. HPLC chromatograms of inosine-modified DNA-Ag16NCs A) monitoring the main absorption peak of the clusters at 530 nm, and B) monitoring the DNA absorption at 260 nm and C) monitoring the emission of the clusters at 730 nm (λexc= 530 nm). The blue, red, and green traces refer, respectively, to I7_DNA-Ag16NC, I9_DNA-Ag16NC and I7-I9_DNA-Ag16NC, whose elution times are reported in section 2.        . Microsecond decay curves of DNA-Ag16NC and inosine mutants synthesized in a 10 mM NH4OAc H2O solution and measured in a 10 mM NH4OAc D2O solution at 25 ⁰C. The decays were monitored at 820 nm, exciting at 508 nm with a Xenon flash lamp (repetition rate = 300 Hz). All decays were tailfitted with a mono-exponential function, and the decay times (<τµs>) are reported in Table 2. The black trace is the IRF, i.e., the instrument response function.             Figure S22. Unit cell of the I7 mutant. Note that 2-Methyl-2,4-pentanediol (yellow compound) cocrystallized. The magenta spheres represent silvers with an occupancy below 1. Figure S23. Unit cell of the I7-I9 mutant comprising six I7-I9_DNA-Ag16NCs. Every subunit shows the same AgNC core, but slightly different bond lengths and positions of the nucleobases. The cross-sections reported in Figure 3 and Figure S9 are from one of these six DNA-AgNCs. The magenta spheres represent silvers with an occupancy below 1.