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

Abstract

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.

Figure 1

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.

Figure 2

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.

Figure 3

Normalized absorption (solid lines) and emission spectra (dashed lines, λ_exc = 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.

Figure 4

(A–D) Chromatograms of compounds 9, 10, 11, and 12 (λ_abs= 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.

Figure 5

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 (λ_exc = 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.