Matthew B. Francis

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.

Integrated microfluidic bioprocessor for single-cell gene expression analysis

An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (≈50%) or complete (≈0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.

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.

High Relaxivity Gadolinium Hydroxypyridonate-Viral Capsid Conjugates: Nano-sized MRI Contrast Agents

High relaxivity macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids are assessed. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a 5-fold increase in relaxivity, leading to a peak relaxivity (per Gd3+ ion) of 41.6 mM(-1) s(-1) at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation to flexible lysines, while internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that (i) there is facile diffusion of water to the interior of capsids and (ii) the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules (q = 2), and the NMRD fittings highlight the differences in the local motion for the internal (tauRl = 440 ps) and external (tauRl = 310 ps) conjugates. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

Chemoselective Tryptophan Labeling with Rhodium Carbenoids at Mild pH

Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6-7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2-7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40-60% conversion with excellent tryptophan chemoselectivity. The solvent accessibility of the indole side chains was found to be a key factor in successful carbenoid addition, as demonstrated by conducting the reaction at temperatures high enough to cause thermal denaturation of the protein substrate. Progress toward the expression of proteins bearing solvent accessible tryptophan residues as reactive handles for modification with rhodium carbenoids is also reported.

Genome-Free Viral Capsids as Multivalent Carriers for Taxol Delivery†

Drugs used in chemotherapy predominantly act by targeting the mechanisms of cell division, 1 and to an extent, preferentially affect cancer cells because of their unusually high proliferation rates. Unfortunately, many healthy cells are also affected by these treatments, resulting in side effects that cause substantial discomfort for the patient. Emerging methods seek to focus the delivery of drugs on cancer tissue by targeting specific characteristics found in solid tumors.2 As a promising subset of these approaches, macromolecular drug delivery seeks to attach many small molecule drugs and targeting groups to large structures.3 Many of these delivery vehicles experience prolonged circulation time because of their increased size,4 as well as a degree of passive targeting through the enhanced permeation and retention effect, arising from unique characteristics of tumor vasculature.5 As an additional benefit, macromolecules are large enough to display multiple copies of active targeting ligands to enhance binding avidity,6 and can also provide multiple cargo attachment sites to increase the amount of payload that can be delivered.

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.

Multivalent, High-Relaxivity MRI Contrast Agents Using Rigid Cysteine-Reactive Gadolinium Complexes

MRI contrast agents providing very high relaxivity values can be obtained through the attachment of multiple gadolinium(III) complexes to the interior surfaces of genome-free viral capsids. In previous studies, the contrast enhancement was predicted to depend on the rigidity of the linker attaching the MRI agents to the protein surface. To test this hypothesis, a new set of Gd-hydroxypyridonate based MRI agents was prepared and attached to genetically introduced cysteine residues through flexible and rigid linkers. Greater contrast enhancements were seen for MRI agents that were attached via rigid linkers, validating the design concept and outlining a path for future improvements of nanoscale MRI contrast agents.

Oxidative coupling of peptides to a virus capsid containing unnatural amino acids.

This Communication describes the chemo- and site-selective coupling of cell type-specific targeting peptides to a virus capsid containing aminophenylalanine residues.

Direct Cell Surface Modification with DNA for the Capture of Primary Cells and the Investigation of Myotube Formation on Defined Patterns

Previously, we reported a method for the attachment of living cells to surfaces through the hybridization of synthetic DNA strands attached to their plasma membrane. The oligonucleotides were introduced using metabolic carbohydrate engineering, which allowed reactive tailoring of the cell surface glycans for chemoselective bioconjugation. While this method is highly effective for cultured mammalian cells, we report here a significant improvement of this technique that allows the direct modification of cell surfaces with NHS-DNA conjugates. This method is rapid and efficient, allowing virtually any mammalian cell to be patterned on surfaces bearing complementary DNA in under 1 h. We demonstrate this technique using several types of cells that are generally incompatible with integrin-targeting approaches, including red blood cells and primary T-cells. Cardiac myoblasts were also captured. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. The availability of this new protocol greatly expands the applicability of the DNA-based attachment strategy for the generation of artificial tissues and the incorporation of living cells into device settings.