Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo.

Selective targeting of cancer cells is a critical step in cancer diagnosis and therapy. To address this need, DNA aptamers have attracted significant attention as possible targeting ligands. However, while their use in targeting cancer cells in vitro has been reported, their effectiveness has rarely been established in vivo. Here we report the development of a liposomal drug delivery system for targeted anticancer chemotherapy. Liposomes were prepared containing doxorubicin as a payload, and functionalized with AS1411, a DNA aptamer with strong binding affinity for nucleolin. AS1411 aptamer-functionalized liposomes increased cellular internalization and cytotoxicity to MCF-7 breast cancer cells as compared to non-targeting liposomes. Furthermore, targeted liposomal doxorubicin improved antitumor efficacy against xenograft MCF-7 breast tumors in athymic nude mice, attributable to their enhanced tumor tissue penetration. This study suggests that AS1411 aptamer-functionalized liposomes can recognize nucleolin overexpressed on MCF-7 cell surface, and therefore enable drug delivery with high specificity and selectivity.

Nano-encrypted Morse code: a versatile approach to programmable and reversible nanoscale assembly and disassembly.

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code "NANO" and reversible exchange of uppercase letter "I" with lowercase "i". The yields of the conjugations are high (>90%), and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.

DNA-directed assembly of asymmetric nanoclusters using Janus nanoparticles.

Asymmetric assembly of nanomaterials has attracted broad interests because of their unique anisotropic properties that are different from those based on the more widely reported symmetric assemblies. Despite the potential advantages, programmable fabrication of asymmetric structure in nanoscale remains a challenge. We report here a DNA-directed approach for the assembly of asymmetric nanoclusters using Janus nanoparticles as building blocks. DNA-functionalized spherical gold nanoparticles (AuNSs) can be selectively attached onto two different hemispheres of DNA-functionalized Janus nanoparticle (JNP) through DNA hybridization. Complementary and invasive DNA strands have been used to control the degree and reversibility of the assembly process through programmable base-pairing interactions, resulting in a series of modular and asymmetric nanostructures that allow systematic study of the size-dependent assembly process. We have also shown that the attachment of the AuNSs onto the gold surface of the Janus nanoparticle results in red shifting of the UV-vis and plasmon resonance spectra.

Controlled alignment of multiple proteins and nanoparticles with nanometer resolution via backbone-modified phosphorothioate DNA and bifunctional linkers.

Controlled alignment of streptavidin (STV), myoglobin, and nanoparticles with nanometer resolution has been achieved via backbone-modified phosphorothioate DNA and biotin- and maleimide-containing bifunctional linkers. Introducing triplet biotin modifications in three adjacent PSs significantly increased the STV conjugation yield. By placing phosphorothioate modifications at multiple positions of a double stranded DNA template, monomer, dimer, and trimer STV-DNA assemblies were formed with the STVs placed at controlled positions. The activity of the conjugated protein has been demonstrated by binding biotinylated AuNPs onto STV-DNA complexes, indicating the use of the system as a template for the formation of AuNP dimers and trimers with STVs separated by distances of 10-30 nm. Furthermore, a melting temperature experiment carried out with an STV-dsDNA assembly showed that the bifunctional-linker-modified PS-DNA system is much more stable than base-modified conjugation systems. This method allows for high yield, nanoscale-precision conjugation of multiple proteins to DNA. The linker can be designed to conjugate any proteins and nanomaterials specifically for a wide range of applications.

Label-free fluorescent functional DNA sensors using unmodified DNA: a vacant site approach.

A general methodology to design label-free fluorescent functional DNA sensors using unmodified DNA via a vacant site approach is described. By extending one end of DNA with a loop, a vacant site that binds an extrinsic fluorophore, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), could be created at a selected position in the DNA duplex region of DNAzymes or aptamers. When the vacant site binds ATMND, ATMND's fluorescence is quenched. This fluorescence can be recovered when one strand of the duplex DNA is released through either metal ion-dependent cleavage by DNAzymes or analyte-dependent structural-switching by aptamers. Through this design, label-free fluorescent sensors for Pb(2+), UO(2)(2+), Hg(2+), and adenosine have been successfully developed. These sensors have high selectivity and sensitivity; detection limits as low as 3 nM, 8 nM, 30 nM, and 6 microM have been achieved for UO(2)(2+), Pb(2+), Hg(2+) and adenosine, respectively. Control experiments using vacant-site-free DNA duplexes and inactive variants of the functional DNAs indicate that the presence of the vacant site and the activity of the functional DNAs are essential for the performance of the proposed sensors. The vacant site approach demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes.

Surface-plasmon-enhanced fluorescence from periodic quantum dot arrays through distance control using biomolecular linkers.

We have developed a protein-enabled strategy to fabricate quantum dot (QD) nanoarrays where up to a 15-fold increase in surface-plasmon-enhanced fluorescence has been achieved. This approach permits a comprehensive control both laterally (via lithographically defined gold nanoarrays) and vertically (via the QD-metal distance) of the collectively behaving assemblies of QDs and gold nanoarrays by way of biomolecular recognition. Specifically, we demonstrated the spectral tuning of plasmon resonant metal nanoarrays and self-assembly of protein-functionalized QDs in a stepwise fashion with a concomitant incremental increase in separation from the metal surface through biotin-streptavidin spacer units.

Arrays of covalently bonded single gold nanoparticles on thiolated molecular assemblies.

A simple approach to form arrays of covalently bonded single gold nanoparticles (AuNPs) is demonstrated. Asymmetric molecular assemblies composed of two layers of rigid aromatic molecules with different structures, arranged in hexagonal arrays on a template produced by edge-spreading lithography, are used to guide the assembly of AuNPs. Arrays of single AuNPs are achieved by taking advantage of the interplay of electrostatic interactions and covalent bonding in conjunction with the positional constraint on the template. Schiff base chemistry is highlighted in the surface chemical reaction to selectively modify nanoscale surface features with high yield.