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DNA-stabilized silver nanoclusters (Ag N -DNAs) are emitters with tuneable structures and photophysical properties. While understanding of the sequence-structure-property relationships of Ag N -DNAs has advanced significantly, their chemical transformations and degradation pathways are far less understood. To advance understanding of these pathways, we analysed the fragmentation products of 21 different red and NIR Ag N -DNAs using negative ion mode electrospray ionization mass spectrometry (ESI-MS). Ag N -DNAs were found to lose Ag + under ESI-MS conditions, and sufficient loss of silver atoms can lead to a transition to a lesser number of effective valence electrons, N 0 . Of more than 400 mass spectral peaks analysed, only even values of N 0 were identified, suggesting that solution-phase Ag N -DNAs with odd values of N 0 are unlikely to be stable. Ag N -DNAs stabilized by three DNA strands were found to fragment significantly more than Ag N -DNAs stabilized by two DNA strands. Moreover, the fragmentation behaviour depends strongly on the DNA template sequence, with diverse fragmentation patterns even for Ag N -DNAs with similar molecular formulae. Molecular dynamics simulations, with forces calculated from density functional theory, of the fragmentation of (DNA) 2 (Ag 16 Cl 2 ) 8+ with a known crystal structure show that the 6-electron Ag 16 Cl 2 core fragments into a 4-electron Ag 10 and a 2-electron Ag 6 , preserving electron-pairing rules even at early stages of the fragmentation process, in agreement with experimental observation. These findings provide new insights into the mechanisms by which Ag N -DNAs degrade and transform, with relevance for their applications in sensing and biomedical applications.

DNA-stabilized silver nanoclusters (Ag N -DNAs) have sequence-tuned compositions and fluorescence colors. High-throughput experiments together with supervised machine learning models have recently enabled design of DNA templates that select for Ag N -DNA properties, including near-infrared (NIR) emission that holds promise for deep tissue bioimaging. However, these existing models do not enable simultaneous selection of multiple Ag N -DNA properties, and require significant expert input for feature engineering and class definitions. This work presents a model for multiobjective, continuous-property design of Ag N -DNAs with automatic feature extraction, based on variational autoencoders (VAEs). This model is generative, i.e., it learns both the forward mapping from DNA sequence to Ag N -DNA properties and the inverse mapping from properties to sequence, and is trained on an experimental data set of DNA sequences paired with Ag N -DNA fluorescence properties. Experimental testing shows that the model enables effective design of Ag N -DNA emission, including bright NIR Ag N -DNAs with 4-fold greater abundance compared to training data. In addition, Shapley analysis is employed to discern learned nucleobase patterns that correspond to fluorescence color and brightness. This generative model can be adapted for a range of biomolecular systems with sequence-dependent properties, enabling precise design of emerging biomolecular nanomaterials.

ConspectusDNA-stabilized silver nanoclusters (Ag N -DNAs) are sequence-encoded fluorophores. Like other noble metal nanoclusters, the optical properties of Ag N -DNAs are dictated by their atomically precise sizes and shapes. What makes Ag N -DNAs unique is that nanocluster size and shape are controlled by nucleobase sequence of the templating DNA oligomer. By choice of DNA sequence, it is possible to synthesize a wide range of Ag N -DNAs with diverse emission colors and other intriguing photophysical properties. Ag N -DNAs hold significant potential as "programmable" emitters for biological imaging due to their combination of small molecular-like sizes, bright and sequence-tuned fluorescence, low toxicities, and cost-effective synthesis. In particular, the potential to extend Ag N -DNAs into the second near-infrared region (NIR-II) is promising for deep tissue imaging, which is a major area of interest for advancing biomedical imaging. Achieving this goal requires a deep understanding of the structure-property relationships that govern Ag N -DNAs in order to design Ag N -DNA emitters with sizes and geometries that support NIR-II emission.In recent years, major advances have been made in understanding the structure and composition of Ag N -DNAs, enabling new insights into the correlation of nanocluster structure and photophysical properties. These advances have hinged on combined innovations in mass characterization and crystallography of compositionally pure Ag N -DNAs, together with combinatorial experiments and machine learning-guided design. A combined approach is essential due to the major challenge of growing suitable Ag N -DNA crystals for diffraction and to the labor-intensive nature of preparing and solving the molecular formulas of atomically precise Ag N -DNAs by mass spectrometry. These approaches alone are not feasibly scaled to explore the large sequence space of DNA oligomer templates for Ag N -DNAs.This account describes recent fundamental advances in Ag N -DNA science that have been enabled by high throughput synthesis and fluorimetry together with detailed analytical studies of purified Ag N -DNAs. First, short introductions to nanocluster chemistry and Ag N -DNA basics are presented. Then, we review recent large-scale studies that have screened thousands of DNA templates for Ag N -DNAs, leading to discovery of distinct classes of these emitters with unique cluster core compositions and ligand chemistries. In particular, the discovery of a new class of chloride-stabilized Ag N -DNAs enabled the first ab initio calculations of Ag N -DNA electronic structure and present new approaches to stabilize these emitters in biologically relevant conditions. Near-infrared (NIR) emissive Ag N -DNAs are also found to exhibit diverse structures and properties. Finally, we conclude by highlighting recent proof-of-principle demonstrations of NIR Ag N -DNAs for targeted fluorescence imaging. Continued efforts may future push Ag N -DNAs into the tissue transparency window for fluorescence imaging in the NIR-II tissue transparency window.

Silver cations can mediate base pairing of guanine (G) DNA oligomers, yielding linear parallel G-Ag + -G duplexes with enhanced stabilities compared to those of canonical DNA duplexes. To enable their use in programmable DNA nanotechnologies, it is critical to understand solution-state formation and the nanomechanical stiffness of G-Ag + -G duplexes. Using temperature-controlled circular dichroism (CD) spectroscopy, we find that heating mixtures of G oligomers and silver salt above 50 °C fully destabilizes G-quadruplex structures and converts oligomers to G-Ag + -G duplexes. Electrospray ionization mass spectrometry supports that G-Ag + -G duplexes form at stoichiometries of 1 Ag + per base pair, and CD spectroscopy suggests that as the Ag + /base stoichiometry increases further, G-Ag + -G duplexes undergo additional morphological changes. Using liquid-phase atomic force microscopy, we find that this excess Ag + enables assembly of long fiberlike structures with ∼2.5 nm heights equivalent to a single DNA duplex but with lengths that far exceed a single duplex. Finally, using the conditions established to form single G-Ag + -G duplexes, we use a surface forces apparatus (SFA) to compare the solution-phase stiffness of single G-Ag + -G duplexes with dG-dC Watson-Crick-Franklin duplexes. SFA shows that G-Ag + -G duplexes are 1.3 times stiffer than dG-dC duplexes, confirming gas-phase ion mobility spectrometry measurements and computational predictions. These findings may guide the development of structural DNA nanotechnologies that rely on silver-mediated base pairing.

We present chemical synthesis strategies for DNA-stabilized silver nanoclusters (Ag N -DNAs) with near-infrared (NIR) emission in the biological tissue transparency windows. Elevated temperatures can significantly increase chemical yield of near-infrared nanoclusters. In most cases, basic pH favors near-infrared nanoclusters while micromolar amounts of NaCl inhibit their formation.

DNA-stabilized silver nanoclusters (Ag N -DNAs) are known to have one or two DNA oligomer ligands per nanocluster. Here, we present the first evidence that Ag N -DNA species can possess additional chloride ligands that lead to increased stability in biologically relevant concentrations of chloride. Mass spectrometry of five chromatographically isolated near-infrared (NIR)-emissive Ag N -DNA species with previously reported X-ray crystal structures determines their molecular formulas to be (DNA) 2 [Ag 16 Cl 2 ] 8+ . Chloride ligands can be exchanged for bromides, which red-shift the optical spectra of these emitters. Density functional theory (DFT) calculations of the 6-electron nanocluster show that the two newly identified chloride ligands were previously assigned as low-occupancy silvers by X-ray crystallography. DFT also confirms the stability of chloride in the crystallographic structure, yields qualitative agreement between computed and measured UV-vis absorption spectra, and provides interpretation of the 35 Cl-nuclear magnetic resonance spectrum of (DNA) 2 [Ag 16 Cl 2 ] 8+ . A reanalysis of the X-ray crystal structure confirms that the two previously assigned low-occupancy silvers are, in fact, chlorides, yielding (DNA) 2 [Ag 16 Cl 2 ] 8+ . Using the unusual stability of (DNA) 2 [Ag 16 Cl 2 ] 8+ in biologically relevant saline solutions as a possible indicator of other chloride-containing Ag N -DNAs, we identified an additional Ag N -DNA with a chloride ligand by high-throughput screening. Inclusion of chlorides on Ag N -DNAs presents a promising new route to expand the diversity of Ag N -DNA structure-property relationships and to imbue these emitters with favorable stability for biophotonics applications.

DNA can stabilize silver nanoclusters (Ag N -DNAs) whose atomic sizes and diverse fluorescence colors are selected by nucleobase sequence. These programmable nanoclusters hold promise for sensing, bioimaging, and nanophononics. However, DNA's vast sequence space challenges the design and discovery of Ag N -DNAs with tailored properties. In particular, Ag N -DNAs with bright near-infrared luminescence above 800 nm remain rare, placing limits on their applications for bioimaging in the tissue transparency windows. Here, we present a design method for near-infrared emissive Ag N -DNAs. By combining high-throughput experimentation and machine learning with fundamental information from Ag N -DNA crystal structures, we distill the salient DNA sequence features that determine Ag N -DNA color, for the entire known spectral range of these nanoclusters. A succinct set of nucleobase staple features are predictive of Ag N -DNA color. By representing DNA sequences in terms of these motifs, our machine learning models increase the design success for near-infrared emissive Ag N -DNAs by 12.3 times as compared to training data, nearly doubling the number of known Ag N -DNAs with bright near-infrared luminescence above 800 nm. These results demonstrate how incorporating known structure-property relationships into machine learning models can enhance materials study and design, even for sparse and imbalanced training data.

DNA oligomers are known to serve as stabilizing ligands for silver nanoclusters (Ag N -DNAs) with rod-like nanocluster geometries and nanosecond-lived fluorescence. Here, we report two Ag N -DNAs that possess distinctly different structural properties and are the first to exhibit only microsecond-lived luminescence. These emitters are characterized by significant broadband downconversion from the ultraviolet/visible to the near-infrared region. Circular dichroism spectroscopy shows that the structures of these two Ag N -DNAs differ significantly from previously reported Ag N -DNAs. We find that these nanoclusters contain eight valence electrons, making them the first reported DNA-stabilized luminescent quasi-spherical superatoms. This work demonstrates the important role that nanocluster composition and geometry play in dictating luminescence properties of Ag N -DNAs and significantly expands the space of structure-property relations that can be achieved for Ag N -DNAs.

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.

The near-infrared (NIR) I and II regions are known for having good light transparency of tissue and less scatter compared to the visible region of the electromagnetic spectrum. However, the number of bright fluorophores in these regions is limited. Here we present a detailed spectroscopic characterization of a DNA-stabilized silver nanocluster (DNA-AgNC) that emits at around 960 nm in solution. The DNA-AgNC converts to blue-shifted emitters over time. Embedding these DNA-AgNCs in poly(vinyl alcohol) (PVA) shows that they are bright and photostable enough to be detected at the single-molecule level. Photon antibunching experiments were performed to confirm single emitter behavior. Our findings highlight that the screening and exploration of DNA-AgNCs in the NIR II region might yield promising bright, photostable emitters that could help develop bioimaging applications with unprecedented signal-to-background ratios and single-molecule sensitivity.

A near-infrared emitting DNA-stabilized silver nanocluster (DNA-AgNC) with an unusually high fluorescence quantum yield is presented. The steady-state and time-resolved fluorescence properties of the DNA-AgNC were characterized, together with its ability to generate optically activated delayed fluorescence (OADF) and upconversion fluorescence (UCF).

Hydrogen bonding between nucleobases produces diverse DNA structural motifs, including canonical duplexes, guanine (G) quadruplexes, and cytosine (C) i-motifs. Incorporating metal-mediated base pairs into nucleic acid structures can introduce new functionalities and enhanced stabilities. Here we demonstrate, using mass spectrometry (MS), ion mobility spectrometry (IMS), and fluorescence resonance energy transfer (FRET), that parallel-stranded structures consisting of up to 20 G-Ag I -G contiguous base pairs are formed when natural DNA sequences are mixed with silver cations in aqueous solution. FRET indicates that duplexes formed by poly(cytosine) strands with 20 contiguous C-Ag I -C base pairs are also parallel. Silver-mediated G-duplexes form preferentially over G-quadruplexes, and the ability of Ag + to convert G-quadruplexes into silver-paired duplexes may provide a new route to manipulating these biologically relevant structures. IMS indicates that G-duplexes are linear and more rigid than B-DNA. DFT calculations were used to propose structures compatible with the IMS experiments. Such inexpensive, defect-free, and soluble DNA-based nanowires open new directions in the design of novel metal-mediated DNA nanotechnology.

We use high throughput near-infrared (NIR) screening technology to discover abundant new DNA-stabilized silver clusters, AgN-DNA, that fluoresce in the NIR. These include the longest wavelength AgN-DNA fluorophores identified to date, with peak emission beyond 950 nm that extends into the NIR II tissue transparency window, and the highest silver content.

DNA nucleobase sequence controls the size of DNA-stabilized silver clusters, leading to their well-known yet little understood sequence-tuned colors. The enormous space of possible DNA sequences for templating clusters has challenged the understanding of how sequence selects cluster properties and has limited the design of applications that employ these clusters. We investigate the genomic role of DNA sequence for fluorescent silver clusters using a data-driven approach. Employing rapid parallel silver cluster synthesis and fluorimetry, we determine the fluorescence spectra of silver cluster products stabilized by 1432 distinct DNA oligomers. By applying pattern recognition algorithms to this large experimental data set, we discover certain DNA base patterns, or "motifs," that correlate to silver clusters with similar fluorescence spectra. These motifs are employed in machine learning classifiers to predictively design DNA template sequences for specific fluorescence color bands. Our method improves selectivity of templates by 330% for silver clusters with peak emission wavelengths beyond 660 nm. The discovered base motifs also provide physical insights into how DNA sequence controls silver cluster size and color. This predictive design approach for color of DNA-stabilized silver clusters exhibits the potential of machine learning and data mining to increase the precision and efficiency of nanomaterials design, even for a soft-matter-inorganic hybrid system characterized by an extremely large parameter space.

In this paper we present a new near-IR emitting silver nanocluster (NIR-DNA-AgNC) with an unusually large Stokes shift between absorption and emission maximum (211 nm or 5600 cm -1 ). We studied the effect of viscosity and temperature on the steady state and time-resolved emission. The time-resolved results on NIR-DNA-AgNC show that the relaxation dynamics slow down significantly with increasing viscosity of the solvent. In high viscosity solution, the spectral relaxation stretches well into the nanosecond scale. As a result of this slow spectral relaxation in high viscosity solutions, a multi-exponential fluorescence decay time behavior is observed, in contrast to the more mono-exponential decay in low viscosity solution.

This work investigates the effects of dielectric environment and cluster shape on electronic excitations of fluorescent DNA-stabilized silver clusters, AgN-DNA. We first establish that the longitudinal plasmon wavelengths predicted by classical Mie-Gans (MG) theory agree with previous quantum calculations for excitation wavelengths of linear silver atom chains, even for clusters of just a few atoms. Application of MG theory to AgN-DNA with 400-850 nm cluster excitation wavelengths indicates that these clusters are characterized by a collective excitation process and suggests effective cluster thicknesses of ∼2 silver atoms and aspect ratios of 1.5 to 5. To investigate sensitivity to the surrounding medium, we measure the wavelength shifts produced by addition of glycerol. These are smaller than reported for much larger gold nanoparticles but easily detectable due to narrower line widths, suggesting that AgN-DNA may have potential for fluorescence-reported changes in dielectric environment at length scales of ∼1 nm.

The diverse optical and chemical properties of DNA-stabilized silver clusters (AgN-DNAs) have challenged the development of a common model for these sequence-tunable fluorophores. Although correlations between cluster geometry and fluorescence color have begun to shed light on how the optical properties of AgN-DNAs are selected, the exact mechanisms responsible for fluorescence remain unknown. To explore these mechanisms, we study four distinct purified AgN-DNAs in ethanol-water and methanol-water mixtures and find that the solvatochromic behavior of AgN-DNAs varies widely among different cluster species and differs markedly from prior results on impure material. Placing AgN-DNAs within the context of standard Lippert-Mataga solvatochromism models based on the Onsager reaction field, we show that such nonspecific solvent models are not universally applicable to AgN-DNAs. Instead, alcohol-induced solvatochromism of AgN-DNAs may be governed by changes in hydration of the DNA template, with spectral shifts resulting from cluster shape changes and/or dielectric changes in the local vicinity of the cluster.

The remarkable precision that DNA scaffolds provide for arraying nanoscale optical elements enables optical phenomena that arise from interactions of metal nanoparticles, dye molecules, and quantum dots placed at nanoscale separations. However, control of ensemble optical properties has been limited by the difficulty of achieving uniform particle sizes and shapes. Ligand-stabilized metal clusters offer a route to atomically precise arrays that combine desirable attributes of both metals and molecules. Exploiting the unique advantages of the cluster regime requires techniques to realize controlled nanoscale placement of select cluster structures. Here we show that atomically monodisperse arrays of fluorescent, DNA-stabilized silver clusters can be realized on a prototypical scaffold, a DNA nanotube, with attachment sites separated by <10 nm. Cluster attachment is mediated by designed DNA linkers that enable isolation of specific clusters prior to assembly on nanotubes and preserve cluster structure and spectral purity after assembly. The modularity of this approach generalizes to silver clusters of diverse sizes and DNA scaffolds of many types. Thus, these silver cluster nano-optical elements, which themselves have colors selected by their particular DNA templating oligomer, bring unique dimensions of control and flexibility to the rapidly expanding field of nano-optics.

We develop approaches to hold fluorescent silver clusters composed of only 10-20 atoms in nanoscale proximity, while retaining the individual structure of each cluster. This is accomplished using DNA clamp assemblies that incorporate a 10 atom silver cluster and a 15 or 16 atom silver cluster. Thermally modulated fluorescence resonance energy transfer (FRET) verifies assembly formation. Comparison to Förster theory, using measured spectral overlaps, indicates that the DNA clamps hold clusters within roughly 5 to 6 nm separations, in the range of the finest resolutions achievable on DNA scaffolds. The absence of spectral shifts in dual-cluster FRET pairs, relative to the individual clusters, shows that select few-atom silver clusters of different sizes are sufficiently stable to retain structural integrity within a single nanoscale DNA construct. The spectral stability of the cluster persists in a FRET pair with an organic dye molecule, in contrast to the blue-shifted emission of the dye.