Yen Nee Tan

Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions.

Gold nanoparticles (AuNPs) are widely used as colorimetric probes for biosensing, relying on their unique particle size-dependent and/or interparticle distance-dependent extinction spectrum and solution color. Herein, we describe an AuNP-based colorimetric assay to detect binding interactions between nuclear hormone receptors and their corresponding DNA-binding elements, particularly the human estrogen receptors (ERalpha and ERbeta) and their cognate estrogen response elements (EREs). We found that the protein-DNA (ER-ERE) complexes can stabilize citrate anion-capped AuNPs against salt-induced aggregation to a larger extent than the protein (ER) or the DNA (ERE) alone, due to their unique molecular size and charge properties that provide a strong electrosteric protection. Moreover, our results show that the extent of stabilization is sequence-dependent and can distinguish a single base variation in the ERE associated with minor changes in protein-DNA binding affinity. With this assay, many important parameters of protein-DNA binding events (e.g., sequence selectivity, distinct DNA binding properties of protein subtypes, binding stoichiometry, and sequence-independent transient binding) can be determined instantly without using labels, tedious sample preparations, and sophisticated instrumentation. These benefits, in particular the high-throughput potential, could enable this assay to become the assay of choice to complement conventional techniques for large scale characterization of protein-DNA interactions, a key aspect in biological research.

Study of single-stranded DNA binding protein-nucleic acids interactions using unmodified gold nanoparticles and its application for detection of single nucleotide polymorphisms.

We have developed a label-free homogeneous phase bioassay to characterize the DNA binding properties of single-stranded DNA binding (SSB) protein, a key protein involved in various DNA processes such as DNA replication and repair. This assay uses gold nanoparticles (AuNPs) as sensing probe and is based on the phenomenon that preformed SSB-single-stranded DNA (ssDNA) complexes can protect AuNPs against salt-induced aggregation better than SSB or ssDNA alone. With the controlled particle aggregation/dispersion as measure, this assay can be used to detect the formation of SSB complexes with ssDNA of different length and nucleotide composition and to assess their binding properties without tedious and complicated assay procedures. On the basis of the inverse relationship between DNA hybridization efficiency and the tendency of SSB to form protection complex with unhybridized ssDNA to AuNPs, this assay is further developed to detect DNA hybridization with single nucleotide polymorphism selectivity. Owing to the high affinity between SSB and dissociated ssDNA, single-base mismatch discrimination in a long sequence of 30-mer DNA was achieved for both the end- and center-base mismatch. Unlike the conventional techniques for DNA and protein analysis, current AuNPs-based sensing strategy is simple in design, fast in detection, and economical for operation without the need of sophisticated equipment.

Sensing of transcription factor through controlled-assembly of metal nanoparticles modified with segmented DNA elements.

We have developed a unique metal nanoparticle (mNPs)-based assay to detect sequence-specific interactions between transcription factor and its corresponding DNA-binding elements. This assay exploits the interparticle-distance dependent optical properties of noble mNPs as sensing element and utilizes specific protein-DNA interactions to control the dispersion status of the mNPs. The assay involves two sets of double-stranded (ds)DNA modified-mNPs, each carrying a half site segment of a functional DNA sequence for the protein of interest. Each of these half sites is designed to contain a short complementary sticky end that introduces base-pairing forces to facilitate particle aggregation and to form a transient full dsDNA sequence. The detection of specific protein-DNA binding is founded on the premise that the mixture of these two sets of dsDNA-mNPs experiences a remarkable particle aggregation under certain salt conditions; whereas the aggregation can be retarded in the presence of a specific protein that binds and stabilizes the transient full dsDNA structure and therefore introduces steric protection forces between particles. We have demonstrated the concept using estrogen receptor α and its response elements, with gold and silver NPs as the sensing platform. UV-vis spectroscopy, transmission electron spectroscopy, and dynamic light scattering measurements were conducted to provide full characterization of the particle aggregation/dispersion mechanism. Differing from most of the mNP-based colorimetric sensors that are designed based on the analyte-induced aggregation mechanism, current protein binding-stabilization sensing strategy reduces the false signals caused by unrelated particle destabilizing effects. It is expected that this assay principle can be directed toward other transcription factors by simply changing the recognition sequence to form different segmented dsDNA-mNP constructs.

Tailoring the protein conformation to synthesize different-sized gold nanoclusters†

Gold nanoclusters with five discrete sizes (Au4, Au8, Au10, Au13, and Au25) are synthesized in a protein template with predefined conformation via a CO-mediated synthesis.

Rational Design of Biomolecular Templates for Synthesizing Multifunctional Noble Metal Nanoclusters toward Personalized Theranostic Applications

Biomolecule-templated or biotemplated metal nanoclusters (NCs) are ultrasmall (<2 nm) metal (Au, Ag) particles stabilized by a certain type of biomolecular template (e.g., peptides, proteins, and DNA). Due to their unique physiochemical properties, biotemplated metal NCs have been widely used in sensing, imaging, delivery and therapy. The overwhelming applications in these individual areas imply the great promise of harnessing biotemplated metal NCs in more advanced biomedical aspects such as theranostics. Although applications of biotemplated metal NCs as theranostic agents are trending, the rational design of biomolecular templates suitable for the synthesis of multifunctional metal NCs for theranostics is comparatively underexplored. This progress report first identifies the essential attributes of biotemplated metal NCs for theranostics by reviewing the state-of-art applications in each of the four modalities of theranostics, namely sensing, imaging, delivery and therapy. To achieve high efficacy in these modalities, we elucidate the design principles underlying the use of biomolecules (proteins, peptides and nucleic acids) to control the NC size, emission color and surface chemistries for post-functionalization of therapeutic moieties. We then propose a unified strategy to engineer biomolecular templates that combine all these modalities to produce multifunctional biotemplated metal NCs that can serve as the next-generation personalized theranostic agents.

DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection.

We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

A highly sensitive fluorescent light-up probe for real-time detection of the endogenous protein target and its antagonism in live cells

Real-time detection and monitoring of cancer-related biomolecular interactions in live cells are of paramount importance for disease diagnostics and drug screening. Herein, we developed a target-specific fluorescent light-up probe for cellular detection of Mdm2, the key negative regulator of the p53 tumour suppressor protein. Conjugation of a uniquely designed fluorogen (TPECM) with aggregation induced-emission properties, to a specific p53-derived peptide (12.1Pep) targeting Mdm2, yielded a cell-permeable probe (TPECM-12.1Pep) with turn-on fluorescence properties for real-time live cell imaging of Mdm2. This specific light-up probe is almost non-fluorescent in its isolated state but is highly emissive upon binding to Mdm2, enabling quantitative detection of both Mdm2 and its antagonism. Using a model compound (Nutlin-3a), we demonstrate that the as-developed probes can be used to screen p53-Mdm2 inhibiting drug candidates, both in vitro and in cells. Furthermore, the probe activity can be accurately monitored in cells using a fluorescently activated cell sorting machine. These features will expedite research in the areas of drug discovery, clinical diagnostics and fundamental cell biology.

Locked Nucleic Acid Gapmers and Conjugates Potently Silence ADAM33, an Asthma-Associated Metalloprotease with Nuclear-Localized mRNA

Two mechanisms dominate the clinical pipeline for oligonucleotide-based gene silencing, namely, the antisense approach that recruits RNase H to cleave target RNA and the RNAi approach that recruits the RISC complex to cleave target RNA. Multiple chemical designs can be used to elicit each pathway. We compare the silencing of the asthma susceptibility gene ADAM33 in MRC-5 lung fibroblasts using four classes of gene silencing agents, two that use each mechanism: traditional duplex small interfering RNAs (siRNAs), single-stranded small interfering RNAs (ss-siRNAs), locked nucleic acid (LNA) gapmer antisense oligonucleotides (ASOs), and novel hexadecyloxypropyl conjugates of the ASOs. Of these designs, the gapmer ASOs emerged as lead compounds for silencing ADAM33 expression: several gapmer ASOs showed subnanomolar potency when transfected with cationic lipid and low micromolar potency with no toxicity when delivered gymnotically. The preferential susceptibility of ADAM33 mRNA to silencing by RNase H may be related to the high degree of nuclear retention observed for this mRNA. Dynamic light scattering data showed that the hexadecyloxypropyl ASO conjugates self-assemble into clusters. These conjugates showed reduced potency relative to unconjugated ASOs unless the lipophilic tail was conjugated to the ASO using a biocleavable linkage. Finally, based on the lead ASOs from (human) MRC-5 cells, we developed a series of homologous ASOs targeting mouse Adam33 with excellent activity. Our work confirms that ASO-based gene silencing of ADAM33 is a useful tool for asthma research and therapy.

Bovine Serum Albulmin Protein‐Templated Silver Nanocluster (BSA‐Ag13): An Effective Singlet Oxygen Generator for Photodynamic Cancer Therapy

This paper reports a novel synthesis approach of bovine serum albumin (BSA) protein-templated ultrasmall (<2 nm) Ag nanocluster (NC) with strong singlet oxygen generation capacity for photodynamic therapy (PDT). An atomically precise BSA-Ag13 NC (i.e., 13 Ag atoms per cluster) is successfully synthesized for the first time by using NaOH-dissolved NaBH4 solution as the controlling reducing agent. The ubiquitous size of BSA-Ag13 NC results in unique behaviors of its photoexcited states as characterized by the ultrafast laser spectroscopy using time-correlated single photon counting and transient absorption techniques. In particular, triply excited states can be largely present in the excited BSA-Ag13 NC and readily sensitized molecular oxygen to produce singlet oxygen (1 O2 ) with a high quantum efficiency (≈1.26 using Rose Bengal as a standard). This value is much higher than its Au analogue (i.e., ≈0.07 for BSA-Au25 NC) and the commonly available photosensitizers. Due to the good cellular uptake and inherent biocompatibility imparted by the surface protein, BSA-Ag13 NC can be applied as an effective PDT agent in generating 1 O2 to kill cancer cell as demonstrated in this study.

A study of DNA design dependency of segmented DNA-induced gold nanoparticle aggregation towards versatile bioassay development

DNA-decorated gold nanoparticles (AuNPs) are important sensing probes for designing versatile assays for detecting DNA hybridization, DNA binders and DNA associated biological processes. In this study, we report DNA design rules for a pair of segmented DNA-conjugated AuNPs that can undergo “transient” aggregation due to cooperative base-pairing force of sticky ends on DNA and salt screening. A wildtype estrogen receptor response element (ERE) was used as a model DNA which is segmented into two half-sites, each carrying a complementary sticky end and a half-site DNA, were conjugated onto AuNPs to form two sets of complementary DNA–AuNPs conjugates. A number of DNA design parameters are studied for their effects on the aggregation kinetics, namely DNA conformation (i.e., mixed structure of ssDNA and dsDNA), number of sticky-ends bases, spacer length, as well as the symmetrical and asymmetrical interplay between the complementary set of segmented DNA–AuNPs conjugates. Firstly, we found that dsDNA serves as a more effective spacer than ssDNA in preventing base coordination of the nucleotides to the AuNPs surface due to its rigidity that in turn helps to improve the accessibility of sticky-ends for faster aggregation. Secondly, base-pairing force in facilitating the salt-induced AuNPs aggregation is tunable by the number of sticky-ends, which is closely related to the length and structural composition of the DNA spacer. Thirdly, symmetrically spaced sticky-ends enable quicker non-crosslinking aggregation than the asymmetrical combination due to their closer initial interparticle distance. Based on the optimized DNA design, we appended the efficient aggregation that leads to transient formation of a full ERE sequence for detecting estrogen receptor β (ER β) by exploiting protein binding retarded particle aggregation. With a competition assay, binding affinity of ERβ to different DNA sequences can be easily screened using a single set of complementary segmented DNA–AuNPs probes. The DNA design dependency of the DNA–AuNPs aggregation studied herein has enabled potential applications for rapid and highly specific DNA-binding protein detection, which could be extended to detect a wide range of DNA binders and its related biological processes.