NanoScript was synthesized by assembling three essential components on AuNPs. The first is a hairpin polyamide structure (representing the DBD), which was specifically chosen because of its high specificity to target DNA sequences and exceptionally high binding affinity that is comparable to naturally occurring DNA-binding proteins.^21,22 The hairpin polyamide is composed of N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids, which bind to complementary A-T and G-C motifs on the DNA, respectively.²³ Using solid-phase synthesis, a polyamide was synthesized with amino acids arranged in the order ImPyPyPy-γ-PyPyPyPy-β-Dp-NH₂ (γ is γ-aminobutyric acid, β is β-alanine, and Dp is dimethylaminopropylamide), to recognize the 5′-WGWWWW-3′ (W = A or T) DNA sequence (Figure 2a). This sequence was specifically chosen to recognize the matching DNA sequence of our proof-of-concept reporter plasmid, which will be used to evaluate its gene-regulating functionality (details described below). Using surface plasmon resonance, the binding affinity of the polyamide to its target sequence was determined to have a remarkably high nanomolar affinity with an equilibrium constant of 1.6 × 10^–9 M (Figure 2b). In addition, the polyamide binding affinity to a mismatched DNA sequence decreased by over 70-fold, thus implying the polyamide is specific for its target sequence (Figure S1).

The second component (representing the AD) is a synthetic transactivation peptide that was synthesized in the d-form to resist intracellular degradation and is capable of activating transcriptional activity by recruiting mediators, RNA polymerase II, SAGA, and other proteins to the binding site.²⁴⁻²⁶ The third component (representing the NLS domain) is a nuclear localization signal peptide derived from the SV40 large T-antigen and functions to shuttle NanoScript into the nucleus.²⁷⁻²⁹

These three components were then assembled onto a single nanoparticle using a carefully designed conjugation chemistry to develop NanoScript. The NanoScript platform was constructed by first coating AuNPs with mercaptoundeconic acid (MUA) and then conjugating the three STF components via EDC/NHS coupling (Figure 3a). This reaction was carried out in a controlled buffered (pH = 6.0–7.4) solution to ensure conjugation through the primary amine, but there is a possibility that NLS binds either directly to the AuNP or through lysine side chains, which should not influence its functionality.²⁹ Adsorption of the STF components on the AuNPs was confirmed by UV spectroscopy, which indicated a successive shift of the surface plasmon peak (Figure S2). On the basis of previous reports, we are confident the DBD conjugated to NanoScript binds preferentially to the target sequence.³⁰ In terms of the STF ratio on NanoScript, we speculated a high ratio of NLS would be required because translocation of NanoScript into the nucleus is one of the most critical barriers for effective gene activation. Furthermore, we anticipated that a minimal ratio of the polyamide DBD would be required because of its exceptionally high binding affinity to DNA and that the ratio of AD be doubled in order to mimic the potent endogenous TF p53.³¹ Therefore, we designed NanoScript to have an optimum ratio of STF components and quantified the surface ratio using high-pressure liquid chromatography (HPLC) analysis (Table S1).

The physical properties were characterized using dynamic light scattering, which determined the hydrodynamic diameter of NanoScript to be 34.0 ± 2.3 nm and the surface charge to be −32.5 mV (Figure 3b). This hydrodynamic diameter is in excellent agreement with the theoretical value, because, based on calculating bond lengths, we predicted the theoretical diameter of NanoScript to be 35.2 nm. Electron micrographs of NanoScript confirmed that surface functionalization with STF components did not affect the size distribution or monodispersity of the nanoparticles (Figure 3c). The stability of both NanoScript and MUA-coated AuNPs (Au-MUA) was tested in various physiological environments including water, PBS, and culture media and showed that the nanoparticles remained stable in these environments, as indicated by minimal shifts in the absorbance peak (Figure S3).

Efficient nuclear localization of NanoScript is especially important because transcriptional activity occurs exclusively in the nucleus. To this end, NanoScript was specifically designed with a small hydrodynamic diameter (∼34.0 nm as mentioned above) because the nuclear pore, which tightly regulates cargo transport across the nuclear envelope, has a maximum diameter of approximately 44 nm.³²⁻³⁴ The plasma membrane permeability of NanoScript was assessed by first incubating NanoScript with HeLa cells for 4 h and then quantifying the number of particles that were able to enter the cells using inductively coupled plasma atomic emission spectroscopy (ICP-OES). Analysis revealed that NanoScript was able to efficiently penetrate the plasma membrane in just 4 h of incubation, as compared to the control (NanoScript without NLS peptide), which showed minimal uptake (Figure 4a), which indicates that the NLS peptide also plays a significant role in enabling NanoScript to enter the cell. To monitor intracellular localization, NanoScript was labeled with a fluorescent dye and incubated in HeLa cell cultures. Fluorescence images, taken with a 3D structured illumination microscope, shows that NanoScript is able to target and penetrate the nuclear envelope (Figure 4b, Figure S4). As indicated by the phase contrast image, the HeLa cells seem to remain intact (Figure S5). Additionally, a side-view image indicates that NanoScript is evenly dispersed throughout the nucleus in the vertical plane, thus confirming that NanoScript is not merely resting on the surface of the nucleus (Figure 4c). Furthermore, a three-dimensional fluorescence video shows NanoScript distributed throughout the nucleus, thereby providing further confirmation of nuclear uptake (Video S1).

Given the potential for degradation of NanoScript components by intracellular proteases, we wanted to ensure that the observed fluorescence overlap was not due to components being cleaved from NanoScript and diffusing into the nucleus, but rather that the nanoparticles were able to enter the nucleus intact. For this purpose, we performed TEM on cellular cross sections to determine if intact NanoScript was able to enter the nucleus. The TEM image shows the interface between the cytoplasm and the nucleus, with NanoScript clearly located inside the nucleus (Figure 4d, Figure S6). While this indicates that the NLS peptide may be initially conjugated on NanoScript, there is high probability that cytoplasmic proteases degrade the NLS peptide over a longer period of time. Combining the results of the fluorescence and TEM images, NanoScript was demonstrated to effectively enter the nucleus while remaining intact, which would be critical for achieving high transcriptional activity.

The primary function of TFs is to regulate transcriptional activity; hence we evaluated NanoScript’s ability to control transcription and gene expression. As a proof-of-concept experiment, we constructed a reporter plasmid containing a response element with six tandem copies of the 5′-TGTTAT-3′ sequence, which is selectively recognized by the polyamide DBD molecule (Figure 5a).¹⁷ This response element is located 36 base pairs upstream from the TATA box to facilitate initiation of transcriptional activity (Figure S7). Transcription of this reporter plasmid produces alkaline phosphatase (ALP), which can be quantified using a colorimetric assay. Since the secreted ALP is directly proportional to the magnitude of induced transcriptional activity, the functionality and potency of NanoScript can be evaluated and quantified. After cotransfecting NanoScript and the reporter plasmid into HeLa cells, ALP levels were measured after 48 h (Figure 5b). Analysis revealed that NanoScript induces transcription and overexpresses the reporter plasmid by 15-fold in comparison to unmodified AuNPs (Figure 5c), while maintaining high cell viability (Figure S8). Furthermore, the contribution of each component on NanoScript for initiating transcriptional activity was tested by selectively removing each component. Subsequent ALP analysis of control experiments showed limited gene expression, thus confirming the importance and synergistic activity of each STF component on NanoScript (Figure 5c). A positive control included transfecting the same concentrations of DBD and AD molecules as on the nanoparticle to the culture media and showed a 2.2-fold increase in gene transcription, a result that is similar to the previously reported studies. Moreover, when cells were exposed to varying NanoScript concentrations, there was dose-dependent transcriptional activation (Figure 5d).

In order to fully emulate the gene-regulating function of TFs and to establish the NanoScript platform suitable for potential biological applications, it is essential that NanoScript can activate endogenous genes on native DNA. For this purpose, several genes including LGALS8 and EGLN3 were identified to contain numerous response elements that complement the polyamide DBD (Table S2).¹⁷ The number of complementary binding sites on endogenous DNA is not expected to be directly proportional to the level of gene activation, primarily due to the nucleosome/histone packaging of DNA and variability in binding site availability.³⁵ Quantification of endogenous gene activation showed that the expression of LGALS8 and EGLN3 was increased 65% and 58%, respectively, relative to unmodified AuNPs (Figure 5e). Successful gene activation indicates that NanoScript can interact with endogenous DNA, seek complementary gene motifs, and initiate transcriptional activity of targeted endogenous genes. It is important to note that the gene activation of the reporter plasmid was expected to be higher than the activation of endogenous genes because endogenous genes are tightly coiled into nucleosomes while reporter plasmids are not, thus making the reporter plasmid more prone to being transcribed. The overexpression of endogenous genes highlights the versatility and functionality of NanoScript, thus proving that this platform can behave and function like natural TFs.

The gold nanoparticles with an approximate diameter of 9 nm were prepared by the Ferns method of citrate reduction of HAuCl₄ following establish protocols. All glassware was cleaned in aqua regia (3:1 HCl/HNO₃, handle with extreme caution!), then rinsed with nanopure water and oven-dried. A 50 mL aqueous solution of 1 mM HAuCl₄ was heated to reflux while stirring. Then 8 mL of 1% (by weight) sodium citrate was quickly added, resulting in a change in solution color from yellow to ruby red. After the color change, the solution was heated to reflux for another 5 min, then cooled to room temperature and filtered using a 0.45 μm syringe filter. Size distribution was characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and concentration was obtained using UV–vis spectroscopy.

Ten nanometer gold nanoparticles were conjugated to ligands using a two-step method. The first step involves ligand exchange on the AuNP with the linker molecule 11-mercaptoundecanoic acid. First, from a stock solution of MUA dissolved in ethanol, 1 mM MUA was added to the AuNP solution (pH = 11, NaOH) and allowed to stir at room temperature for 24 h. The solution was then filtered three times using a 10 000 MWCO filter (Millipore) and resuspended in distilled water. The second step involves conjugating the ligands to the AuNP via the carboxylic acid of the MUA. To the AuNP solution, adjusted to pH = 6.5 by adding 5 mM MES (Hampton Research), was added 0.3 mM 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide) (EDC) (Sigma) and 0.75 mM N-hydroxysuccinimide (NHS) (Acros Organics), and the resulting solution was allowed to stir for 2 h at room temperature. The solution was filtered three times using a 10K MWCOF and resuspended in 1 mM MES water. Immediately afterward, a solution containing a 10 M excess of NLS peptide, transactivation peptide (TAP), and hairpin-polyamide DBD with a mole ratio of 7:2:1 (NLS:TAP:DBD) was added dropwise to the AuNP solution, adjusted to pH = 7.2 by adding 50 mM HEPES (Cellgro) prior to addition of biomolecules, and allowed to stir for 2 h. The AuNP solution was filtered three times using a 10 000 MWCO to remove unreacted molecules.

The dye-labeled NanoScript, used for tracking intracellular localization, was constructed by modifying the AD (the transactivation peptide) with a fluorescent dye (Alexa Fluor 568 Hydrazide, Invitrogen). Specifically, the dye was conjugated to the transactivation peptide via EDC/NHS coupling as described above.

The NanoScript construct was characterized using multiple techniques. First, the concentration of the gold nanoparticles and confirmation of conjugation were found using UV–visible absorption spectra (Varian Cary 5000 UV–vis–NIR spectrophotometer). Second, DLS (Malvern Zetasizer Nano-ZS90) was utilized to measure the size and zeta potential (surface charge) of the AuNP construct after each conjugation step to find the hydrodynamic diameter and surface charge, respectively. Third, the morphology of the AuNP core was determined using TEM analysis. The AuNPs were drop-cast on the holey-carbon grids (Electron Microscopy Sciences), allowed to dry overnight under vacuum, and subsequently imaged using a JEOL JEM-2010F high-resolution TEM operated at an accelerating voltage of 200 kV. Finally, the amount of peptides on NanoScript’s surface was calculated by performing HPLC (Agilent LC 1100) using a Zorbax Extend-C18 Solvent Saver Plus, 3.5 μm, 3.0 × 150 mm, column. The peptide solution before conjugation and the supernatant solution after conjugation, which contained unreacted peptides, were both analyzed using HPLC. The exact number of moles for each peptide was calculated using a standard curve comparing the concentration and area. The difference in moles for each peptide before and after conjugation was calculated. The ratio of the difference in moles is attributed to the ratio on the nanoparticle. For this experiment, after the nanoparticles were centrifuged at 12 000 rpm for 20 min, the supernatant was collected and analyzed.

HeLa cells were cultured in Dulbecco’s modified Eagle medium with high glucose (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum, 1% Glutamax (Invitrogen), and 1% streptomycin–penicillin antibiotic in a 37 °C humidified incubator with 5% CO₂. Prior to transfection, 25 000 HeLa cells were seeded in a well of a 24-well plate. After 24 h, the cells were incubated with a mixture of the 1 nM NanoScript solution, 0.5 μg of the reporter plasmid, and X-tremeGENE (Roche) in Opti-MEM medium (Invitrogen). After 4 h, the cells were washed twice with PBS and fresh culture medium was added. After 48 h, the alkaline phosphatase level in the culture medium was tested using the SEAP chemiluminescence kit 2.0 (Clontech) following the manufacturer’s protocol. An MTS assay (Promega) was also performed at this time to test cell viability.