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. 2023 Jul 13;145(30):16771–16777. doi: 10.1021/jacs.3c04768

Bioconjugation of a Near-Infrared DNA-Stabilized Silver Nanocluster to Peptides and Human Insulin by Copper-Free Click Chemistry

Vanessa Rück †, Narendra K Mishra ‡, Kasper K Sørensen ‡, Mikkel B Liisberg †, Ane B Sloth §,∥, Cecilia Cerretani †, Christian B Mollerup ⊥, Andreas Kjaer §,∥, Chenguang Lou #, Knud J Jensen ‡, Tom Vosch †,*
PMCID: PMC10402711  PMID: 37441791

Abstract

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DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

Introduction

DNA-stabilized silver nanoclusters (DNA-AgNCs) were first introduced by Petty et al. in 2004,1 and have become known for their tunable emission, high brightness, large Stokes shift, and interesting photophysical properties.2 Usually, less than 30 silver atoms and cations are embedded in one or more DNA strands with an overall diameter below 2 nm.2 The DNA oligomer acts both as a scaffold to prevent aggregation into bigger particles and as a programmable tool to generate atomically precise clusters.3−8 Hence, tuning the DNA template results in a wide palette of DNA-AgNCs with emission spanning from the visible to the near-infrared (NIR) range.2 Due to the large variability provided by the four natural nucleotides alone, the photophysical properties of DNA-AgNCs can be modified in a myriad of different ways.2,9 Additionally, simple modifications of the nucleobases, e.g., replacing guanosine with inosine, whose structures solely differ in one amino group, can dramatically affect the fluorescence decay time and quantum yield, as was recently demonstrated.10−13 DNA-AgNCs are interesting not only for unraveling and understanding the origin of luminescence in small metal clusters but also for their use as fluorophores. Most DNA-AgNCs possess fluorescent states along with microsecond-lived states, which can be either dark or luminescent. The presence of the long-lived state enables the possibility of optically activated delayed fluorescence (OADF) and upconversion fluorescence (UCF).14,15 Since the emission is on the anti-Stokes side of the secondary excitation laser, these approaches allow background-free imaging. Moreover, the high brightness of some clusters in the near-infrared region16 makes them promising candidates for imaging biological samples, e.g., tissues and cells, characterized by high autofluorescence. However, limited work has been done on attaching functional groups to DNA-AgNCs in order to promote conjugation to targets of interest,17 which is key to improving their potential as fluorophores in bioimaging applications.18−21 A likely problem is that the linker can influence the interactions of the nucleobases with the AgNC, leading to changes in the spectroscopic properties or loss of stability of the DNA-AgNC. Here, we demonstrate a bioconjugation strategy for an NIR-emitting DNA-AgNC with a known structure.22 Our approach is based on the copper-free click reaction achieved by strain-promoted azido-alkyne cycloaddition (SPAAC).23−25 The spontaneous addition of a reactive, ring-strained alkyne to an azido group eliminates the need for a metal catalyst like copper, which is cytotoxic.24

Particularly, we prove the successful conjugation of the NIR emissive DNA-Ag16NC22,26,27 to three different peptides of various sizes as well as a small protein, human insulin (hI). For hI conjugated to DNA-Ag16NC, confocal fluorescence microscopy was performed to visualize human insulin receptor B (hIR-B) overexpressed in Chinese hamster ovary (CHO) cells. In addition, emission spectra and fluorescence lifetime images were recorded to prove that the photophysical properties of DNA-Ag16NCs are not affected by the cellular environment, a problem (e.g., quenching or spectral shifts) that can occur with other fluorophores.28 To the best of our knowledge, this is the first time that SPAAC has been used to successfully conjugate biomolecules to purified DNA-AgNCs.

Results and Discussion

Synthesis of BCN-Terminated DNA-Ag16NC

To evaluate our proposed strategy, the well-characterized DNA-Ag16NC was chosen. This specific DNA-AgNC was selected for several reasons. First, it is an NIR emitter with a very large Stokes shift (5600 cm–1)26,29 and has a decent quantum yield (0.26)26 compared to other fluorophores in the same emission range.30 Moreover, it was recently found that DNA-Ag16NC has a red-shifted μs-lived state, which could be used to generate OADF, opening up the possibility of time-gated background-free imaging.27 Additionally, it could be used as a suitable two-photon fluorophore for measuring blood flow velocities in living mice.31 Most importantly, the structure is known,22 which is crucial in the rational design of our conjugation experiments. Crystallographic data show that the AgNC is well-protected by the DNA template, and thus it displays remarkable chemical stability over time.

The DNA-Ag16NC is formed by two 10-base DNA sequences 5′-CACCTAGCGA-3′.22 Based on the structure, the 5′-ends are not available for conjugation, since the cytosines are coordinated by Ag atoms. The attachment of a functional group in this position, as well as one of the internal positions, could affect the formation of the AgNC and alter the spectroscopic properties. In contrast, the A10 nucleotides at the 3′-ends do not interact with the AgNC.22 Since the removal of A10 was proven not to affect the photophysical properties of Ag16NC,32 the 3′-end position was selected for attaching a reactive linking group.

For the copper-free click reaction, a ring-strained alkyne is required; thus, we opted for bicyclononyne (BCN), which is the smallest ring-strained alkyne that can be easily dissolved in water. BCN enables highly efficient and bio-orthogonal conjugation to peptides and proteins when exposed to an azide-containing counterpart.23−25 In order to attach a BCN group to the 3′-end of 5′-CACCTAGCGA-3′, the DNA was ordered with a primary amine bound to the A10 position (compound 1, see Figure 1).

Figure 1.

Figure 1

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Scheme for the synthesis and conjugation of DNA-BCN-Ag16NC (4) with three peptides and a small protein: PYY-peptide (5), d-peptide (6), coiled-coil peptide (7), and human insulin (8). 9, 10, 11, and 12 are the conjugated products of compound 4 to peptides 5, 6, 7 and protein 8, respectively. The DNA-Ag16NC structure was made in Pymol using PDB accession code 6JR4.22

The first step was the synthesis of compound 3 (step A in Figure 1). Compound 1, dissolved in phosphate-buffered saline (PBS) and triethylamine, was mixed with BCN-NHS-ester (compound 2) dissolved in acetonitrile (see Supporting Information (SI) for further details). The BCN-terminated DNA (compound 3) was then purified by spin-filtration. Liquid chromatography mass spectrometry (LCMS) showed that the desired compound 3 was obtained (Figure S1). Afterward, compound 3 was used to prepare the BCN-terminated DNA-Ag16NC (compound 4, step B in Figure 1). Briefly, compound 3 was mixed with AgNO3 in 10 mM ammonium acetate (NH4OAc), to which a freshly prepared NaBH4 solution was added after 15 min to promote the formation of the AgNC. The final ratio between the components was [DNA]:[AgNO3]:[NaBH4] = 25 μM:187.5 μM:93.75 μM, based on a previously published protocol.26

After synthesis, the reaction mixture was stored in the refrigerator for 1–3 days prior to high-performance liquid chromatography (HPLC) purification. Details on the HPLC method and the corresponding chromatograms can be found in the SI. Finally, the purified fraction was solvent exchanged with 10 mM NH4OAc by spin-filtration. As can be seen in Figure 2A, a single peak is present in the LCMS chromatogram, which indicates that mainly compound 4 was obtained, and no byproducts were present. However, compound 4 highly fragmented with the applied LCMS conditions (see Figure S3). Even though this is common for DNA-AgNCs,1,33,34 Gonzàlez-Rosell et al. have recently managed to obtain the mass spectrum of DNA-Ag16NC and unraveled that the chemical structure of the silver cluster is [Ag16Cl2]8+.35 Using similar experimental conditions,35 compound 4 was further analyzed by a different mass spectrometer, which enabled us to confirm that the DNA-BCN-Ag16NC was successfully synthesized. The mass spectrum is reported in Figure 2B, where z = 5– and 4– peaks are highlighted. These peaks correspond to the mass of DNA-Ag16NC with two BCN groups attached (Figure 2C). Interestingly, additional peaks corresponding to DNA-BCN-AgNC with a larger number of silver atoms could also be observed in the mass spectrum. These extra silver ions are not part of the AgNC core, but are most likely coordinated to the triple bond of the BCN group.36 A detailed explanation of why we believe these additional silver cations are coordinated to the triple bond and how this affects the MS measurement can be found in the SI.

Figure 2.

Figure 2

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LCMS data for compound 4. (A) Chromatogram monitoring the absorbance at 254 nm, given in mOD. Compound 4 elutes at approximately 2.4 min. See Figure S3 for the corresponding mass spectrum. (B) Mass spectrum of compound 4 measured on a different mass spectrometer (see SI). The stars indicate the peaks related to compound 4 with 16 silver atoms. (C) Zoomed-in views of the marked peaks in the mass spectrum for z = 5– and 4– peaks, along with the corresponding Gaussian fits and average molecular masses (μ). See SI for further details on why the mass ratio is off by approximately 2 amu.

Spectroscopic characterization of compound 4 showed that the photophysical properties are unaltered by the attachment of the BCN groups (Table 1 and Figures 3, S20–S21). The absorption and emission maxima are at 524 and 743 nm, respectively, and the average fluorescence decay time ⟨τ⟩ remains at around 3.26 ns. Time-resolved fluorescence anisotropy measurements were used to determine the hydrodynamic volume of compound 4 and all conjugates. Compared to DNA-Ag16NC alone (10.14 nm3), the hydrodynamic volume of compound 4 increased, as expected, to 12.18 nm3, due to the addition of the two BCN groups (Table 1 and Figure 1).

Table 1. Steady-State and Time-Resolved Fluorescence Data, Along with Time-Resolved Anisotropy Data of DNA-Ag16NC, DNA-BCN-Ag16NC (Compound 4) and DNA-BCN-Ag16NC Conjugated to Three Peptides and a Protein (Compounds 9–12)a.

Compound λabs (nm) λem (nm) ⟨τ⟩ (ns) r1 θ1 (ns) r2 θ2 (ns) Vhydro1 (nm3) Vhydro2 (nm3)
DNA-Ag16NC 526 746 3.22 0.388 2.19 – – 10.14 –
4 524 743 3.26 0.382 2.63 – – 12.18 –
9 525 738 3.07 0.117 2.63* 0.246 6.77 12.18 31.35
10 525 740 3.07 0.233 2.63* 0.143 7.06 12.18 32.66
11 523 741 3.24 0.322 2.63* 0.0548 3.28 12.18 15.18
12 526 734 3.10 0.174 2.63* 0.207 8.60 12.18 39.80
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a

λabs and λem are the absorption and emission maxima, ⟨τ⟩ is the intensity-weighted average decay time monitored at 740 nm (λexc = 507.5 nm), r is the fundamental anisotropy, θ is the rotational correlation time, and Vhydro is the calculated hydrodynamic volume. The subscripts 1 and 2 refer to the two components derived from the biexponential fit of the anisotropy data measured at 740 nm, exciting at 507.5 nm (see SI and Figures S20–S25 for further details on the spectroscopic measurements). *This value was fixed based on the rotational correlation time of compound 4.

Figure 3.

Figure 3

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Normalized absorption (solid lines) and emission spectra (dashed lines, λexc = 507.5 nm) of the original DNA-Ag16NC and the DNA-BCN-Ag16NC (compound 4) in 10 mM NH4OAc at room temperature. See Figure S20 for the full absorption spectrum of compound 4.

Conjugation of Compound 4 to Peptides and hI

After compound 4 was successfully synthesized, we proceeded with the copper-free click reactions to three azido-modified peptides (PYY-peptide 5, d-peptide 6, coiled-coil peptide 7)37−39 and a small azido-modified protein, human insulin (compound 8),40 as shown in step C of Figure 1. The copper-free click reaction of compound 4 with each peptide was carried out at 18 °C by shaking the mixture at 350 rpm for 72 h. Analytical HPLC and LCMS analysis confirmed that each reaction was completed after 72 h (more details can be found in the SI). Several temperatures and reaction times were tested, and these reaction conditions were found to give the highest yields. The LCMS chromatograms of compounds 9, 10, and 11 are shown in Figure 4A–C, where mainly single peaks are present, while the corresponding mass spectra can be found in Figures S13–15. Compounds 9–11 retained the color of the original DNA-Ag16NC solution, confirming that DNA-Ag16NC is stable under the applied reaction conditions. This observation was further verified by spectroscopic characterization. Figure S22 shows that absorption and emission spectra of compounds 9–11 remain unchanged compared to compound 4 (see also Table 1). Reaction of the peptides with DNA-BCN-Ag16NC was also proven by time-resolved anisotropy measurements. Unlike the original DNA-Ag16NC and compound 4, the time-resolved anisotropy data of compounds 9–11 could be best fitted with two rotational correlation times. The first rotational correlation time (θ1) was fixed in the fit to the value of compound 4, representing the local motion of the DNA-Ag16NC part, while the second component (θ2) was left free and was significantly longer, representing the rotation of the entire construct (Table 1).

Figure 4.

Figure 4

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(A–D) Chromatograms of compounds 9, 10, 11, and 12 (λabs= 254 nm), respectively. The absorbance is given in mOD. The corresponding mass spectra can be found in Figures S13–15 and S18. All four compounds have a long shelf life; see Figure S28.

The latter indicates that the overall hydrodynamic volume increased due to conjugation with the peptides. However, we could not confirm with certainty that the value of θ2 matched with the addition of the peptides. First, the error in the calculated hydrodynamic volume is large when the rotational correlation time is significantly longer than the fluorescence decay time. Second, no hydrodynamic volumes for the three peptides could be found in literature.

Next, we applied the click reaction to a therapeutically relevant protein, human insulin. This well-studied protein is essential in the regulation of glucose metabolism and diabetes treatment. The copper-free click reaction was carried out in water by adding compound 8 to compound 4. The reaction mixture was shaken at 850 rpm for 36 h at 37 °C. LCMS analysis confirmed that the reaction was successful, as could be seen from the single peak around 3.1 min (see Figures 4D and S18). Absorption and emission spectra of compound 12 remained unchanged compared to the DNA-BCN-Ag16NC (see Figure S24). Also for compound 12, two rotational correlation times were needed to satisfactorily fit the time-resolved anisotropy data. The first rotational correlation time (θ1) represented the movement of the DNA-BCN-Ag16NC part, while the second component (θ2) was significantly longer (Table 1). The hydrodynamic volume calculated from θ2 was found to be 39.80 nm3. The monomer of bovine insulin, which is similar in size to human insulin, has been reported to have a hydrodynamic radius of 1.4 nm.41 For a spherical approximation, this yields a volume of 11.50 nm3. Even though the combined hydrodynamic volume is not a simple addition, the value of 39.80 nm3 for compound 12 seems to indicate that DNA-BCN-Ag16NC reacts with two hI units.

Bioimaging of hI-Conjugated DNA-Ag16NC

Fluorescence imaging studies were carried out on Chinese hamster ovary (CHO) cells with hI-conjugated DNA-Ag16NC (compound 12) to demonstrate that our linking strategy provides a straightforward labeling protocol and makes DNA-Ag16NCs suitable for bioimaging applications. Prior to bioimaging studies, the chemical stability of compound 4 in the cell medium was tested by recording emission spectra over time. Figure S26 displays a negligible drop in the emission intensity, confirming the good chemical stability of DNA-BCN-Ag16NCs with no decomposition of the compound and potential release of silver. Therefore, we can assume that the insulin conjugate (compound 12) shows similar behavior since the stability of the DNA-Ag16NC is most likely the critical part of the construct. Afterward, the photostability of compound 4 in the cell medium was also verified by continuously irradiating the sample for 2 h (excitation power = 1.67 mW yielding approximately 0.1 W/cm2). As shown in Figure S27, compound 4 has good photostability, since only a minor decrease in the fluorescence intensity can be observed.

For the fluorescence imaging experiments, CHO cells overexpressing human insulin receptor B (hIR-B) were used.42 The CHO cells were first fixed, incubated with compound 12 or DNA-Ag16NC (as control) for 15 min, and then imaged in both bright field and confocal fluorescence microscopy configurations (see SI for details).

Figure 5A–B shows that compound 12 was mostly localized on the cell membrane. To verify that the observed emission is not due to autofluorescence, emission spectra were recorded from the stained regions. Figure 5E confirms that the recorded fluorescence in Figure 5B is indeed from insulin-conjugated DNA-Ag16NCs, as the expected Gaussian-like emission band centered at 730 nm was obtained. It should be noted that upon increasing the concentration of compound 12, the labeling was no longer limited to the outer membrane but covered the entire cell (Figure S29). This concentration-dependent staining of CHO cells is in line with previously reported findings by Ghosh et al.43 To exclude the possibility that some of the staining was due to nonspecific interactions of DNA-Ag16NCs with the fixed CHO cells, similar experiments were performed with DNA-Ag16NCs alone (Figure 5C–D). As shown by the blue spectrum in Figure 5E, no significant emission could be detected when using similar staining concentrations and imaging conditions. All DNA-Ag16NCs were clearly removed during the washing step before imaging (see SI for more details). Consequently, only autofluorescence, similar to unstained CHO cells, was detected in the control experiment (see Figure S30). Additionally, fluorescence lifetime imaging microscopy (FLIM) was performed, and the recorded images and data can be found in Figures 5F–G and S31. The fluorescence decays acquired for every pixel were fitted with a biexponential reconvolution function, including the autogenerated instrument response function (IRF). The mean value of 2.93 ns from the Gaussian fit distribution of the fluorescence decay times is very close to that obtained from bulk measurements (3.10 ns, see Table 1), indicating that compound 12 was not significantly affected by the cellular environment. Furthermore, compound 12 was localized on the CHO cell membranes, in agreement with the confocal fluorescence intensity image in Figure 5B.

Figure 5.

Figure 5

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Imaging of fixed CHO cells overexpressing hIR-B receptors. (A) Bright-field and (B) confocal images of CHO cells stained with approximately 100 nM compound 12. (C) Bright-field and (D) confocal images of CHO cells stained with approximately 100 nM DNA-Ag16NCs. (E) Emission spectra (λexc = 520 nm) of CHO cells stained with compound 12 (orange curve) and DNA-Ag16NCs only (light blue trace). (F) FLIM image of CHO cells overexpressing hIR-B labeled with approximately 300 nM DNA-Ag16NC-hI (compound 12) and (G) corresponding histogram of fitted fluorescence decay times. A Gaussian fit to the distribution yields a mean value (μ) of 2.93 ns. Scale bars: 10 μm.

In the end, a cell viability assay was carried out to evaluate the applicability of DNA-Ag16NCs to living cells. CHO cells were incubated with increasing concentrations (ranging from 50 nM to 1 μM) of DNA-Ag16NC and compound 12 along with human insulin (azido-hI) as a control. After 4 h, approximately 90% of the cells were still viable when treated with concentrations up to 500 nM. On the other hand, a 30% drop was recorded for cells incubated with a 1 μM concentration of DNA-Ag16NC and compound 12, while human insulin did not significantly affect the cell viability (see SI and Figure S32 for further details). Overall the concentrations used for the bioimaging experiments (100−300 nM) do not appear to be toxic for living cells.

Conclusions

We demonstrated successful copper-free click reactions between NIR emissive DNA-Ag16NCs and peptides of different sizes as well as a small protein. The described approach focused on the rational design of DNA-BCN-Ag16NC, based on the addition of the BCN group at the 3′-end of the original sequence. As proven by the mass spectrometry data, the presence of the linkers does not affect the formation of DNA-Ag16NC, and the photophysical properties remain unaltered. The conjugated compounds were analyzed by MS, HPLC, and time-resolved anisotropy measurements. As shown for the human insulin adduct, we were able to image CHO cells with DNA-Ag16NCs by achieving good and specific staining of the membranes. Control experiments excluded the possibility of nonspecific binding. Furthermore, spectral and lifetime measurements confirmed that the spectroscopic properties of DNA-Ag16NCs were preserved in biologically relevant environments.

The described site-specific conjugation of DNA-AgNCs via SPAAC is a promising approach to generate fluorescent labels from DNA-AgNCs once their structures become available. This avoids tedious testing of the DNA sequence positions in order to find a suitable nucleobase where the linker can be attached. Particularly, the DNA-AgNC should have at least a noninteracting nucleobase in the stabilizing DNA strand to make it suitable for the click reaction, while retaining the original photophysical properties. The proposed strategy can open up new application possibilities and hopefully stimulate other research groups to use this new class of emitters to address biologically relevant problems.

Acknowledgments

V.R., M.B.L., C.C., and T.V. acknowledge funding from the Villum Foundation (VKR023115) and the Independent Research Fund Denmark (0136-00024B). N.K.M., K.K.S., and K.J.J. thank the Villum Foundation (VKR18333) for funding the Biomolecular Nanoscale Engineering Center (BioNEC), a VILLUM center of excellence, and the Novo Nordisk Foundation funding for the Center for Biopharmaceuticals and Biobarriers in Drug Delivery (BioDelivery; Grand Challenge Program; NNF16OC0021948). The authors would like to thank Morten Lundh from Gubra for providing the CHO cells expressing the hIR-B.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c04768.

  • Material and Methods section, experimental details on the synthesis, LCMS, MS, HPLC purification and spectroscopic characterization, as well as staining procedure and confocal imaging. (PDF)

The authors declare no competing financial interest.

Supplementary Material

ja3c04768_si_001.pdf (15.9MB, pdf)

References

  1. Petty J. T.; Zheng J.; Hud N. V.; Dickson R. M. DNA-Templated Ag Nanocluster Formation. J. Am. Chem. Soc. 2004, 126, 5207–5212. 10.1021/ja031931o. [DOI] [PubMed] [Google Scholar]
  2. Gonzàlez-Rosell A.; Cerretani C.; Mastracco P.; Vosch T.; Copp S. M. Structure and luminescence of DNA-templated silver clusters. Nanoscale Advances 2021, 3, 1230–1260. 10.1039/D0NA01005G. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Copp S. M.; Gorovits A.; Swasey S. M.; Gudibandi S.; Bogdanov P.; Gwinn E. G. Fluorescence color by data-driven design of genomic silver clusters. ACS Nano 2018, 12, 8240–8247. 10.1021/acsnano.8b03404. [DOI] [PubMed] [Google Scholar]
  4. Copp S. M.; Swasey S. M.; Gorovits A.; Bogdanov P.; Gwinn E. G. General Approach for Machine Learning-Aided Design of DNA-Stabilized Silver Clusters. Chem. Mater. 2020, 32, 430–437. 10.1021/acs.chemmater.9b04040. [DOI] [Google Scholar]
  5. Sharma J.; Yeh H.-C.; Yoo H.; Werner J. H.; Martinez J. S. Silver nanocluster aptamers: in situ generation of intrinsically fluorescent recognition ligands for protein detection. Chem. Commun. 2011, 47, 2294–2296. 10.1039/C0CC03711G. [DOI] [PubMed] [Google Scholar]
  6. Yeh H.-C.; Sharma J.; Han J. J.; Martinez J. S.; Werner J. H. A DNA-Silver Nanocluster Probe That Fluoresces upon Hybridization. Nano Lett. 2010, 10, 3106–3110. 10.1021/nl101773c. [DOI] [PubMed] [Google Scholar]
  7. Petty J. T.; Giri B.; Miller I. C.; Nicholson D. A.; Sergev O. O.; Banks T. M.; Story S. P. Silver Clusters as Both Chromophoric Reporters and DNA Ligands. Anal. Chem. 2013, 85, 2183–2190. 10.1021/ac303531y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Latorre A.; Somoza A. DNA-Mediated Silver Nanoclusters: Synthesis, Properties and Applications. ChemBioChem. 2012, 13, 951–958. 10.1002/cbic.201200053. [DOI] [PubMed] [Google Scholar]
  9. Gonzàlez-Rosell A.; Guha R.; Cerretani C.; Rück V.; Liisberg M. B.; Katz B. B.; Vosch T.; Copp S. M. DNA Stabilizes Eight-Electron Superatom Silver Nanoclusters with Broadband Downconversion and Microsecond-Lived Luminescence. J. Phys. Chem. Lett. 2022, 13, 8305–8311. 10.1021/acs.jpclett.2c02207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Zhang Y.; He C.; de La Harpe K.; Goodwin P. M.; Petty J. T.; Kohler B. A single nucleobase tunes nonradiative decay in a DNA-bound silver cluster. J. Chem. Phys. 2021, 155, 094305. 10.1063/5.0056836. [DOI] [PubMed] [Google Scholar]
  11. Zhang Y.; He C.; Petty J. T.; Kohler B. Time-Resolved Vibrational Fingerprints for Two Silver Cluster-DNA Fluorophores. J. Phys. Chem. Lett. 2020, 11, 8958–8963. 10.1021/acs.jpclett.0c02486. [DOI] [PubMed] [Google Scholar]
  12. Petty J. T.; Ganguly M.; Yunus A. I.; He C.; Goodwin P. M.; Lu Y.-H.; Dickson R. M. A DNA-Encapsulated Silver Cluster and the Roles of Its Nucleobase Ligands. J. Phys. Chem. C 2018, 122, 28382–28392. 10.1021/acs.jpcc.8b09414. [DOI] [Google Scholar]
  13. Cerretani C.; Liisberg M.; Rück V.; Kondo J.; Vosch T. The effect of inosine on the spectroscopic properties and crystal structure of a NIR-emitting DNA-stabilized silver nanocluster. Nanoscale Advances 2022, 4, 3212–3217. 10.1039/D2NA00325B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fleischer B. C.; Petty J. T.; Hsiang J. C.; Dickson R. M. Optically Activated Delayed Fluorescence. J. Phys. Chem. Lett. 2017, 8, 3536–3543. 10.1021/acs.jpclett.7b01215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krause S.; Carro-Temboury M. R.; Cerretani C.; Vosch T. Anti-Stokes fluorescence microscopy using direct and indirect dark state formation. Chem. Commun. 2018, 54, 4569–4572. 10.1039/C8CC01521J. [DOI] [PubMed] [Google Scholar]
  16. Neacşu V. A.; Cerretani C.; Liisberg M. B.; Swasey S. M.; Gwinn E. G.; Copp S. M.; Vosch T. Unusually large fluorescence quantum yield for a near-infrared emitting DNA-stabilized silver nanocluster. Chem. Commun. 2020, 56, 6384–6387. 10.1039/D0CC01849J. [DOI] [PubMed] [Google Scholar]
  17. Choi S.; Dickson R. M.; Yu J. Developing luminescent silver nanodots for biological applications. Chem. Soc. Rev. 2012, 41, 1867–1891. 10.1039/C1CS15226B. [DOI] [PubMed] [Google Scholar]
  18. Antoku Y.; Hotta J. I.; Mizuno H.; Dickson R. M.; Hofkens J.; Vosch T. Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters. Photochemical and Photobiological Sciences 2010, 9, 716–721. 10.1039/c0pp00015a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yu J.; Choi S.; Dickson R. M. Shuttle-Based Fluorogenic Silver-Cluster Biolabels. Angew. Chem., Int. Ed. 2009, 48, 318–320. 10.1002/anie.200804137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Richards C. I.; Hsiang J. C.; Senapati D.; Patel S.; Yu J.; Vosch T.; Dickson R. M. Optically modulated fluorophores for selective fluorescence signal recovery. J. Am. Chem. Soc. 2009, 131, 4619–4621. 10.1021/ja809785s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Choi S.; Yu J.; Patel S. A.; Tzeng Y.-L.; Dickson R. M. Tailoring silver nanodots for intracellular staining. Photochemical & Photobiological Sciences 2011, 10, 109–115. 10.1039/c0pp00263a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Cerretani C.; Kanazawa H.; Vosch T.; Kondo J. Crystal structure of a NIR-Emitting DNA-Stabilized Ag16 Nanocluster. Angewandte Chemie - International Edition 2019, 58, 17153–17157. 10.1002/anie.201906766. [DOI] [PubMed] [Google Scholar]
  23. Agard N. J.; Prescher J. A.; Bertozzi C. R. A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126, 15046–15047. 10.1021/ja044996f. [DOI] [PubMed] [Google Scholar]
  24. Kim E.; Koo H. Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo. Chemical Science 2019, 10, 7835–7851. 10.1039/C9SC03368H. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Dommerholt J.; Schmidt S.; Temming R.; Hendriks L. J. A.; Rutjes F. P. J. T.; van Hest J. C. M.; Lefeber D. J.; Friedl P.; van Delft F. L. Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells. Angew. Chem., Int. Ed. 2010, 49, 9422–9425. 10.1002/anie.201003761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Bogh S. A.; Carro-Temboury M. R.; Cerretani C.; Swasey S. M.; Copp S. M.; Gwinn E. G.; Vosch T. Unusually large Stokes shift for a near-infrared emitting DNA-stabilized silver nanocluster. Methods and Applications in Fluorescence 2018, 6, 024004. 10.1088/2050-6120/aaa8bc. [DOI] [PubMed] [Google Scholar]
  27. Liisberg M.; Krause S.; Cerretani C.; Vosch T. Probing Emission of a DNA-Stabilized Silver Nanocluster from the Sub-Nanosecond to Millisecond Timescale in a Single Measurement. Chemical Science 2022, 13, 5582–5587. 10.1039/D2SC01137A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lakowicz J. R.Principles of fluorescence spectroscopy; Springer: 2006. [Google Scholar]
  29. Chen J.; Kumar A.; Cerretani C.; Vosch T.; Zigmantas D.; Thyrhaug E. Excited-State Dynamics in a DNA-Stabilized Ag16 Cluster with Near-Infrared Emission. J. Phys. Chem. Lett. 2023, 14, 4078–4083. 10.1021/acs.jpclett.3c00764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. East A. K.; Lucero M. Y.; Chan J. New directions of activity-based sensing for in vivo NIR imaging. Chemical Science 2021, 12, 3393–3405. 10.1039/D0SC03096A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wang X.; Liisberg M. B.; Nolt G. L.; Fu X.; Cerretani C.; Li L.; Johnson L. A.; Vosch T.; Richards C. I. DNA-AgNC Loaded Liposomes for Measuring Cerebral Blood Flow Using Two-Photon Fluorescence Correlation Spectroscopy. ACS Nano 2023, 10.1021/acsnano.3c04489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Cerretani C.; Kondo J.; Vosch T. Removal of the A10 adenosine in a DNA-stabilized Ag16 nanocluster. RSC Adv. 2020, 10, 23854–23860. 10.1039/D0RA02672G. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vosch T.; Antoku Y.; Hsiang J. C.; Richards C. I.; Gonzalez J. I.; Dickson R. M. Strongly emissive individual DNA-encapsulated Ag nanoclusters as single-molecule fluorophores. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12616–12621. 10.1073/pnas.0610677104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Gwinn E. G.; O’Neill P.; Guerrero A. J.; Bouwmeester D.; Fygenson D. K. Sequence-Dependent Fluorescence of DNA-Hosted Silver Nanoclusters. Adv. Mater. 2008, 20, 279–283. 10.1002/adma.200702380. [DOI] [Google Scholar]
  35. Gonzàlez-Rosell A.; Malola S.; Guha R.; Arevalos N. R.; Rafik M.; Matus M. F.; Goulet M. E.; Haapaniemi E.; Katz B. B.; Vosch T.; Kondo J.; Häkkinen H.; Copp S. M. Chloride ligands on DNA-stabilized silver nanoclusters. J. Am. Chem. Soc. 2023, 145, 10721–10729. 10.1021/jacs.3c01366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Fang G.; Bi X. Silver-catalysed reactions of alkynes: recent advances. Chem. Soc. Rev. 2015, 44, 8124–8173. 10.1039/C5CS00027K. [DOI] [PubMed] [Google Scholar]
  37. Beck-Sickinger A. G.; Jung G. Structure-activity relationships of neuropeptide Y analogues with respect to Y1 and Y2 receptors. Biopolymers 1995, 37, 123–142. 10.1002/bip.360370207. [DOI] [PubMed] [Google Scholar]
  38. Lou C.; Boesen J. T.; Christensen N. J.; Sørensen K. K.; Thulstrup P. W.; Pedersen M. N.; Giralt E.; Jensen K. J.; Wengel J. Self-Assembly of DNA-Peptide Supermolecules: Coiled-Coil Peptide Structures Templated by d-DNA and l-DNA Triplexes Exhibit Chirality-Independent but Orientation-Dependent Stabilizing Cooperativity. Chem.—Eur. J. 2020, 26, 5676–5684. 10.1002/chem.201905636. [DOI] [PubMed] [Google Scholar]
  39. Ljubetic A.; Lapenta F.; Gradisar H.; Drobnak I.; Aupic J.; Strmsek Z.; Lainscek D.; Hafner-Bratkovic I.; Majerle A.; Krivec N.; Bencina M.; Pisanski T.; Velickovic T. C.; Round A.; Carazo J. M.; Melero R.; Jerala R. Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo. Nat. Biotechnol. 2017, 35, 1094–1101. 10.1038/nbt.3994. [DOI] [PubMed] [Google Scholar]
  40. Østergaard M.; Mishra N. K.; Jensen K. J. The ABC of Insulin: The Organic Chemistry of a Small Protein. Chem.—Eur. J. 2020, 26, 8341–8357. 10.1002/chem.202000337. [DOI] [PubMed] [Google Scholar]
  41. Wang P.; Wang X.; Liu L.; Zhao H.; Qi W.; He M. The Hydration Shell of Monomeric and Dimeric Insulin Studied by Terahertz Time-Domain Spectroscopy. Biophys. J. 2019, 117, 533–541. 10.1016/j.bpj.2019.06.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mannerstedt K.; Mishra N. K.; Engholm E.; Lundh M.; Madsen C. S.; Pedersen P. J.; Le-Huu P.; Pedersen S. L.; Buch-Månson N.; Borgström B.; Brimert T.; Fink L. N.; Fosgerau K.; Vrang N.; Jensen K. J. An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins. Chem.—Eur. J. 2021, 27, 3166–3176. 10.1002/chem.202004878. [DOI] [PubMed] [Google Scholar]
  43. Ghosh S.; Chattoraj S.; Bhattacharyya K. Solvation Dynamics and Intermittent Oscillation of Cell Membrane: Live Chinese Hamster Ovary Cell. J. Phys. Chem. B 2014, 118, 2949–2956. 10.1021/jp412631d. [DOI] [PubMed] [Google Scholar]

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