(A to C) NovoTag designs for the binding of JF494 (A), JF596 (B), and JF657 (C). For each JF dye–NovoTag pair, the chemical structure of the target dye (left), model of the NovoTag design (middle) with the relevant dye-interacting residues highlighted (insets), and fluorescence polarization (FP) titration curves (right) are shown. For each FP titration, 3-8 independent measurements were acquired. For each concentration, the FP (mean ± SD) is shown. A non-linear regression (standard binding isotherm model) was fitted, and Kd values were determined and reported with standard error (SE). (D) Excitation (dotted lines) and emission (solid lines) of JF dye-NovoTag pairs: JF494 (green), JF596 (orange), and JF657 (red). (E) Photophysical characterization of free JF dyes and JF dye–NovoTag conjugates: Absorbance maximum (λabs), emission maximum (λem), extinction coefficient (ε), fluorescence quantum yield (Φf) with SD, and fluorescence lifetime (τf). (F and G) Superposition of the design model of NovoTag657 (red) with the apo (F) and holo (G) crystal structure (gray). Green dashed lines indicate hydrogen bonds.#

(A and B) Multiplexed fluorescence microscopy in fixed (A) and live (B) HeLa cells: NovoTag494 labels endosomes (2×FYVE, magenta), NovoTag596 labels mitochondria (MitoTag, green), and NovoTag657 labels chromatin (H2B, white). Cells were stained with 50 nM of each dye (JF494, JF596, JF657) for 30 min at 37°C, followed by three washing steps. Confocal and STED images (presented side by side at the top) were acquired on a Leica Stellaris 8 STED Falcon microscope. Bottom: numbered zoom-ins and line plots (ii, iv) for NovoTag494 (iii) and NovoTag596 (iv) for confocal and STED. (C) Photostability of JF dyes: HeLa cells expressing either NovoTag657 or HaloTag7 localized to mitochondria (MitoTag) were fluorescently labeled with 50 nM of each dye (JF657, Halo-JFX650) as above. Fluorescence intensity was measured after each bleaching iteration. For each sample, photobleaching of 10 individual cells was acquired in two independent experiments. All data points are shown (gray). Non-linear regression (one-phase decay) of the fluorescence signal was fitted. (D) Specificity of NovoTags: Each NovoTag (NovoTag494, NovoTag596, NovoTag657) was localized to mitochondria (MitoTag) in HeLa cells, labeled with one of each JF dye (JF494, JF596, JF657), and excited with one of three wavelengths (474 nm, 575 nm, 631 nm). Fluorescence intensity measurements were obtained from twelve fields of view for each condition in three independent experiments. For each combination, the mean fluorescence intensity is plotted. Scale bars in (A) and (B), 5 μm, zoom 1-2: 5 μm, zoom 3-6: 500 nm.#

除了改变光谱波长外，研究团队还探索了利用蛋白质工程对荧光寿命进行精准微调的可能性。如图3所示，研究人员通过对NovoTag494结合口袋的氢键网络和静电环境进行序列重设计，筛选出了短荧光寿命变体NovoTag494S（1.27 ns）与长荧光寿命变体NovoTag494L（2.78 ns），与原始设计（2.00 ns）形成了显著的分离。在细胞实验中，利用单一波长激发和相位荧光寿命解离技术（FLIM），研究团队仅凭一种JF494染料便成功区分了内体、线粒体和染色质三个不同的细胞器结构，将多色成像拓展到了荧光寿命的全新维度。

(A) NovoTag494 binding site was subjected to sequence redesign using LigandMPNN-FastRelax, and two variants with different hydrogen-bonding networks, π–π-stacking profiles, and electrostatics of the binding pocket were selected with shorter or longer fluorescence lifetimes (NovoTag494S and NovoTag494L). The sequence logo represents the relative frequency (letter height) of each amino acid in the redesigned residues. (B and C) Excitation (B) and emission (C) of JF494 dye bound to NovoTag494 and the two lifetime variants. (D) Photophysical characterization of the JF494-NovoTag494 variant conjugates. Parameters as specified in Fig. 1D. (E) Fluorescence polarization titration of NovoTag494, and of the two lifetime variants. A nonlinear regression (standard binding isotherm model) was fitted, and the Kd values were determined. NovoTag494S and NovoTag494L showed affinities to JF494 of 49.8 nM and 150.8 nM, respectively. For each sample, 3-4 independent measurements were acquired. (F) Distribution of fluorescence lifetimes of each NovoTag494 variant in cells. HeLa cells were transfected with one of the following plasmids: MitoTag-NovoTag494S, 2×FYVE-NovoTag494, or H2B-NovoTag494L, chemically fixed, and imaged in the presence of 10 nM JF494. For each sample, fluorescence images of 11-16 cellular regions were acquired. The histograms of fluorescence lifetimes are plotted, and fitted with a Gaussian distribution. (G to J) HeLa cells were co-transfected with MitoTag-NovoTag494S, 2×FYVE-NovoTag494, and H2B-NovoTag494L, chemically fixed, and imaged in the presence of 10 nM JF494 with a confocal microscope Leica Stellaris 8 Falcon. Shown are the total fluorescence intensity (G), the fluorescence lifetimes (H), and the phasor plot (I). Using phasor-based lifetime unmixing, the signals of the three labeled components were separated (J). Scale bar: 20 μm.#

为了进一步拓展应用场景，研究团队还开发了共价结合与诱导二聚化系统。通过在结合口袋特定位置引入半胱氨酸残基，成功构建了能与JF657发生亲核取代反应的共价标签NovoTag657cv，在活细胞内实现了稳定的共价标记。如图4所示，研究人员将NovoTag657切分为两个片段，构建了名为NovoSplit657的化学诱导二聚化系统。在活细胞中，加入JF657染料可迅速诱导两个蛋白片段组装成二聚体，同时提供强烈的荧光定位信号。更令人瞩目的是，当在细胞固定后加入染料时，NovoSplit657能够作为极低背景的近距离探针，精准记录固定前细胞内天然蛋白质的相互作用状态，而不会引入人工诱导的非特异性聚集。

(A) The design of NovoSplit657 is based on NovoTag657 that was split at two sites, and the original C- and N-termini were fused with a short linker. The fluorescent dye JF657 induces dimerization of the two monomers NovoSplit657A and NovoSplit657B. To prevent non-induced dimerization, an Arg53Ala substitution was introduced into NovoSplit657B. (B) The NovoSplit657 AF3 prediction (in color) superimposed with the crystal structure of NovoTag657 (gray). (C to E) NovoSplit657 as a CID system: HeLa cells were transfected with MitoTag-mStayGold-NovoSplit657A and mCherry-NovoSplit657B (C). Cells were incubated with 200 nM JF657 for 1 hour, washed three times, chemically fixed, and imaged with a confocal microscope Zeiss LSM 980 AiryScan (D). Colocalization was analyzed by calculating the Pearson’s correlation coefficient between NovoSplit657A and NovoSplit657B, as well as between NovoSplit657A and JF657 (E). For each sample, 59 (non-induced) and 61 (+JF657) individual cells were analyzed. (F to J) NovoSplit657 as proximity biosensor: HeLa cells were transfected with MitoTag-LHD(A)-NovoSplit657A-mScarlet and either LHD(B)-NovoSplit657B-mNeonGreen (F) or NovoSplit657B-mNeonGreen (negative control, H). Cells were chemically fixed and fluorescently labeled with 5 nM JF657 for 30 min, followed by three washing steps. Fluorescence images were acquired on an OMX SR microscope [(G) and (I)]. For experiment in (G), colocalization was analyzed by calculating the Pearson’s correlation coefficient between NovoSplit657A and NovoSplit657B, as well as between NovoSplit657A and JF657 (J). For each sample, 11 (LHD(A)-LHD(B)) and 12 (LHD(A)) individual cells were analyzed. All data are shown as Box and Whiskers plots indicating the median (center of box), 25% and 75% quartiles (bounds of box), minimum and maximum values (bars). Each data point represents one cell. Unpaired two-sided Welch’s t tests were conducted. Statistical significance: ∗∗∗∗ p < 0.0001. Scale bars: (D) 20 μm, (G) and (I) 40 μm.#