Nicholas Stephanopoulos

Choosing an effective protein bioconjugation strategy

The collection of chemical techniques that can be used to attach synthetic groups to proteins has expanded substantially in recent years. Each of these approaches allows new protein targets to be addressed, leading to advances in biological understanding, new protein-drug conjugates, targeted medical imaging agents and hybrid materials with complex functions. The protein modification reactions in current use vary widely in their inherent site selectivity, overall yields and functional group compatibility. Some are more amenable to large-scale bioconjugate production, and a number of techniques can be used to label a single protein in a complex biological mixture. This review examines the way in which experimental circumstances influence ones selection of an appropriate protein modification strategy. It also provides a simple decision tree that can narrow down the possibilities in many instances. The review concludes with example studies that examine how this decision process has been applied in different contexts.

Dual-Surface Modified Virus Capsids for Targeted Delivery of Photodynamic Agents to Cancer Cells

Bacteriophage MS2 was used to construct a targeted, multivalent photodynamic therapy vehicle for the treatment of Jurkat leukemia T cells. The self-assembling spherical virus capsid was modified on the interior surface with up to 180 porphyrins capable of generating cytotoxic singlet oxygen upon illumination. The exterior of the capsid was modified with ∼20 copies of a Jurkat-specific aptamer using an oxidative coupling reaction targeting an unnatural amino acid. The capsids were able to target and selectively kill more than 76% of the Jurkat cells after only 20 min of illumination. Capsids modified with a control DNA strand did not target Jurkat cells, and capsids modified with the aptamer were found to be specific for Jurkat cells over U266 cells (a control B cell line). The doubly modified capsids were also able to kill Jurkat cells selectively even when mixed with erythrocytes, suggesting the possibility of using our system to target blood-borne cancers or other pathogens in the blood supply.

Immobilization and One-Dimensional Arrangement of Virus Capsids with Nanoscale Precision Using DNA Origami

DNA origami was used as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. To do this, we first modified the interior surface of bacteriophage MS2 capsids with fluorescent dyes as a model cargo. An unnatural amino acid on the external surface was then coupled to DNA strands that were complementary to those extending from origami tiles. Two different geometries of DNA tiles (rectangular and triangular) were used. The capsids associated with tiles of both geometries with virtually 100% efficiency under mild annealing conditions, and the location of capsid immobilization on the tile could be controlled by the position of the probe strands. The rectangular tiles and capsids could then be arranged into one-dimensional arrays by adding DNA strands linking the corners of the tiles. The resulting structures consisted of multiple capsids with even spacing (approximately 100 nm). We also used a second set of tiles that had probe strands at both ends, resulting in a one-dimensional array of alternating capsids and tiles. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multicomponent systems from biological scaffolds using the power of rationally engineered DNA nanostructures.

Impact of Assembly State on the Defect Tolerance of TMV-Based Light Harvesting Arrays

Self-assembling, light harvesting arrays of organic chromophores can be templated using the tobacco mosaic virus coat protein (TMVP). The efficiency of energy transfer within systems containing a high ratio of donors to acceptors shows a strong dependence on the TMVP assembly state. Rod and disk assemblies derived from a single stock of chromophore-labeled protein exhibit drastically different levels of energy transfer, with rods significantly outperforming disks. The origin of the superior transfer efficiency was probed through the controlled introduction of photoinactive conjugates into the assemblies. The efficiency of the rods showed a linear dependence on the proportion of deactivated chromophores, suggesting the availability of redundant energy transfer pathways that can circumvent defect sites. Similar disk-based systems were markedly less efficient at all defect levels. To examine these differences further, the brightness of donor-only systems was measured as a function of defect incorporation. In rod assemblies, the photophysical properties of the donor chromophores showed a significant dependence on the number of defects. These differences can be partly attributed to vertical energy transfer events in rods that occur more rapidly than the horizontal transfers in disks. Using these geometries and the previously measured energy transfer rates, computational models were developed to understand this behavior in more detail and to guide the optimization of future systems. These simulations have revealed that significant differences in excited state dissipation rates likely also contribute to the greater efficiency of the rods and that statistical variations in the assembly process play a more minor role.

Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons

We report the construction of DNA nanotubes covalently functionalized with the cell adhesion peptide RGDS as a bioactive substrate for neural stem cell differentiation. Alteration of the Watson–Crick base pairing program that builds the nanostructures allowed us to probe independently the effect of nanotube architecture and peptide bioactivity on stem cell differentiation. We found that both factors instruct synergistically the preferential differentiation of the cells into neurons rather than astrocytes.

Dramatic thermal stability of virus-polymer conjugates in hydrophobic solvents.

We have developed a method for integrating the self-assembling tobacco mosaic virus capsid into hydrophobic solvents and hydrophobic polymers. The capsid was modified at tyrosine residues to display an array of linear poly(ethylene glycol) chains, allowing it to be transferred into chloroform. In a subsequent step, the capsids could be transferred to a variety of hydrophobic solvents, including benzyl alcohol, o-dichlorobenzene, and diglyme. The thermal stability of the material against denaturation increased from 70 °C in water to at least 160 °C in hydrophobic solvents. With a view toward material fabrication, the polymer-coated TMV rods were also incorporated into solid polystyrene and thermally cast at 110 °C. Overall, this process significantly expands the range of processing conditions for TMV-based materials, with the goal of incorporating these templated nanoscale systems into conductive polymer matrices.

Antibody-guided photoablation of voltage-gated potassium currents

A family of 40 mammalian voltage-gated potassium (Kv) channels control membrane excitability in electrically excitable cells. The contribution of individual Kv channel types to electrophysiological signaling has been difficult to assign, as few selective inhibitors exist for individual Kv subunits. Guided by the exquisite selectivity of immune system interactions, we find potential for antibody conjugates as selective Kv inhibitors. Here, functionally benign anti-Kv channel monoclonal antibodies (mAbs) were chemically modified to facilitate photoablation of K currents. Antibodies were conjugated to porphyrin compounds that upon photostimulation inflict localized oxidative damage. Anti-Kv4.2 mAb–porphyrin conjugates facilitated photoablation of Kv4.2 currents. The degree of K current ablation was dependent on photon dose and conjugate concentration. Kv channel photoablation was selective for Kv4.2 over Kv4.3 or Kv2.1, yielding specificity not present in existing neurotoxins or other Kv channel inhibitors. We conclude that antibody–porphyrin conjugates are capable of selective photoablation of Kv currents. These findings demonstrate that subtype-specific mAbs that in themselves do not modulate ion channel function are capable of delivering functional payloads to specific ion channel targets.

Instructing cells with programmable peptide DNA hybrids

The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed.

Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles

The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.

Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals.

The foundational goal of structural DNA nanotechnology-the field that uses oligonucleotides as a molecular building block for the programmable self-assembly of nanostructured systems-was to use DNA to construct three-dimensional (3D) lattices for solving macromolecular structures. The programmable nature of DNA makes it an ideal system for rationally constructing self-assembled crystals and immobilizing guest molecules in a repeating 3D array through their specific stereospatial interactions with the scaffold. In this work, we have extended a previously described motif (4 × 5) by expanding the structure to a system that links four double-helical layers; we use a central weaving oligonucleotide containing a sequence of four six-base repeats (4 × 6), forming a matrix of layers that are organized and dictated by a series of Holliday junctions. In addition, we have assembled mirror image crystals (l-DNA) with the identical sequence that are completely resistant to nucleases. Bromine and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for both forms and solution of the resulting structures to 3.0 and 3.05 Å resolution. Both right- and left-handed forms crystallized in the trigonal space groups with mirror image 3-fold helical screw axes P32 and P31 for each motif, respectively. The structures reveal a highly organized array of discrete and well-defined cavities that are suitable for hosting guest molecules and allow us to dictate a priori the assembly of guest-DNA conjugates with a specified crystalline hand.