Large-scale investigation of the effects of nucleobase sequence on fluorescence excitation and Stokes shifts of DNA-stabilized silver clusters

Abstract

DNA-stabilized silver clusters (AgN-DNAs) exhibit diverse sequence-programmed fluorescence, making these tunable nanoclusters promising sensors and bioimaging probes. Recent advances in the understanding of AgN-DNA structures and optical properties have largely relied on detailed characterization of single species isolated by chromatography. Because most AgN-DNAs are unstable under chromatography, such studies do not fully capture the diversity of these clusters. As an alternative method, we use high-throughput synthesis and spectroscopy to measure steady state Stokes shifts of hundreds of AgN-DNAs. Steady state Stokes shift is of interest because its magnitude is determined by energy relaxation processes which may be sensitive to specific cluster geometry, attachment to the DNA template, and structural engagement of solvent molecules. We identify 305 AgN-DNA samples with single-peaked emission and excitation spectra, a characteristic of pure solutions and single emitters, which thus likely contain a dominant emissive AgN-DNA species. Steady state Stokes shifts of these samples vary widely, are in agreement with values reported for purified clusters, and are several times larger than for typical organic dyes. We then examine how DNA sequence selects AgN-DNA excitation energies and Stokes shifts, comment on possible mechanisms for energy relaxation processes in AgN-DNAs, and discuss how differences in AgN-DNA structure and DNA conformation may result in the wide distribution of optical properties observed here. These results may aid computational studies seeking to understand the fluorescence process in AgN-DNAs and the relations of this process to AgN-DNA structure.

Figure 1:

Prototypical excitation (dashed black line) and emission (solid black line) spectra for an Ag_N-DNA with single excitation and emission peaks. The emission spectrum is fitted to a single Gaussian (red shading) to determine peak emission energy E_em. The excitation spectrum is fitted to two single Gaussian peaks, one in the visible-NIR region at fitted peak energy E_ex (green shaded fit in Region 1) and one in the UV (purple shaded fit in Region 2). Signal between 306-330 nm is excluded due to stray light in some regions of the well plate from an imperfection in the plate reader. Stokes shift SS is calculated as SS = E_ex − E_em. The ratio of excitation peak areas in Regions 1 and 2 is defined as R_UV/vis = (area of green Region 2 peak)/(area of purple Region 1 peak). Figure S7 shows additional excitation spectra of samples excluded from analysis due to multiple peaks in excitation spectra.

Figure 2:

a) E_em versus E_ex for purified Ag_N-DNAs (cyan squares; data from previous studies^,,,,,) and the spectrally pure Ag_N-DNAs identified here (data points with error bars; colors correlate to Stokes shift magnitude as indicated by upper left color bar). Error bars represent standard deviations of Gaussian least squares fits. Dashed black line indicates E_em = E_ex, (SS = 0). Solid green lines are equivalent to the upper spectral wavelength limits of this study. b-c) Histograms of b) E_ex and c) E_em of single-peaked Ag_N-DNA (black bars). Fourteen colored vertical lines represent b) E_ex and c) E_em of purified Ag_N-DNAs with N₀ = 4 (green), N₀ = 6 (red), and N₀ = 10 to 12 (blue), as determined by MS.^,,,

Figure 3:

a) SS versus E_ex for spectrally pure solutions of Ag_N-DNAs (black), previously characterized HPLC-purified Ag_N-DNAs (cyan squares),^,,,,, and organic fluorophores which are commonly used to label oligonucleotides (red circles). Vertical and horizontal error bars of black points represent standard deviations; other markers are larger in size than associated standard deviations. b) Heatmap of the data for spectrally pure Ag_N-DNAs from (a) illustrates relative abundance of certain pairs of (E_ex, SS) (legend indicates color meaning).

Figure 4:

R_UV/vis, ratio of areas of UV excitation peak (Region 2) to excitation peak (Region 1), as a function of a) E_ex, b) SS, and c) extinction coefficient of the template DNA strand, calculated using the nearest neighbor model.^,

Figure 5:

Mean values of (a) E_ex, (b) E_em, (c) SS, and (d) R_UV/vis for each instance of the four nucleobases and the 16 possible two-base motifs in the set of DNA template sequences for the 305 spectrally pure Ag_N-DNAs. Error bars represent standard error, as a measure of precision of the mean.

Figure 6:

a) Indication of low SS (green) and high SS (orange) categories, separated at the median value of SS = 0.336 eV. b, c) Motifs are sorted left to right in order of greatest relative difference between orange and green bars.