Predicting monoclonal antibody binding sequences from a sparse sampling of all possible sequences.

Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences.

Modeling the sequence dependence of differential antibody binding in the immune response to infectious disease.

Past studies have shown that incubation of human serum samples on high density peptide arrays followed by measurement of total antibody bound to each peptide sequence allows detection and discrimination of humoral immune responses to a variety of infectious diseases. This is true even though these arrays consist of peptides with near-random amino acid sequences that were not designed to mimic biological antigens. This "immunosignature" approach, is based on a statistical evaluation of the binding pattern for each sample but it ignores the information contained in the amino acid sequences that the antibodies are binding to.

Two-Dimensional Excitonic Networks Directed by DNA Templates as an Efficient Model Light-Harvesting and Energy Transfer System.

Photosynthetic organisms organize discrete light-harvesting complexes into large-scale networks to facilitate efficient light collection and utilization. Inspired by nature, herein, synthetic DNA templates were used to direct the formation of dye aggregates with a cyanine dye, K21, into discrete branched photonic complexes, and two-dimensional (2D) excitonic networks. The DNA templates ranged from four-arm DNA tiles, ≈10 nm in each arm, to 2D wireframe DNA origami nanostructures with different geometries and varying dimensions up to 100×100 nm.

Comprehensive Prediction of Molecular Recognition in a Combinatorial Chemical Space Using Machine Learning.

In combinatorial chemical approaches, optimizing the composition and arrangement of building blocks toward a particular function has been done using a number of methods, including high throughput molecular screening, molecular evolution, and computational prescreening. Here, a different approach is considered that uses sparse measurements of library molecules as the input to a machine learning algorithm which generates a comprehensive, quantitative relationship between covalent molecular structure and function that can then be used to predict the function of any molecule in the possible combinatorial space. To test the feasibility of the approach, a defined combinatorial chemical space consisting of ∼10 possible linear combinations of 16 different amino acids was used.

Application of multiplexed pharmacokinetic immunoassay to quantify drug forms and coadministered biologics.

Meso Scale Discovery U-PLEX provides an opportunity to develop multiplexed pharmacokinetic (PK) immunoassays. Two case studies demonstrate the utility of multiplexed PK methods. Development of PK ligand-binding assays quantify of nonclinical plasma concentrations of a biotherapeutic that has degraded due to biotransformation, and clinical serum concentrations from two biotherapeutics spiked into a single sample.

Efficient Long-Range, Directional Energy Transfer through DNA-Templated Dye Aggregates.

The benzothiazole cyanine dye K21 forms dye aggregates on double-stranded DNA (dsDNA) templates. These aggregates exhibit a red-shifted absorption band, enhanced fluorescence emission, and an increased fluorescence lifetime, all indicating strong excitonic coupling among the dye molecules. K21 aggregate formation on dsDNA is only weakly sequence dependent, providing a flexible approach that is adaptable to many different DNA nanostructures.

Directed Energy Transfer through DNA-Templated J-Aggregates.

Strongly coupled molecular dye aggregates have unique optoelectronic properties that often resemble those of light harvesting complexes found in Nature. The exciton dynamics in coupled dye aggregates could enhance the long-range transfer of optical excitation energy with high efficiency. In principle, dye aggregates could serve as important components in molecular-scale photonic devices; however, rational design of these coupled dye aggregates with precise control over their organization, interactions, and dynamics remains a challenge.

Influence of the Electrochemical Properties of the Bacteriochlorophyll Dimer on Triplet Energy-Transfer Dynamics in Bacterial Reaction Centers.

Energetics, protein dynamics, and electronic coupling are the key factors in controlling both electron and energy transfer in photosynthetic bacterial reaction centers (RCs). Here, we examine the rates and mechanistic pathways of the PH radical-pair charge recombination, triplet state formation, and subsequent triplet energy transfer from the triplet state of the bacteriochlorophyll dimer (P) to the carotenoid in a series of mutant RCs (L131LH + M160LH (D1), L131LH + M197FH (D2), and L131LH + M160LH + M197FH (T1)) of Rhodobacter sphaeroides. In these mutants, the electronic structure of P is perturbed and the P/P midpoint potential is systematically increased due to addition of hydrogen bonds between P and the introduced residues.

Peptide Sequencing Directly on Solid Surfaces Using MALDI Mass Spectrometry.

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications.

Programmed coherent coupling in a synthetic DNA-based excitonic circuit.

Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer.

Enhancing Photocurrent Generation in Photosynthetic Reaction Center-Based Photoelectrochemical Cells with Biomimetic DNA Antenna.

Three- to four-times higher performance of biohybrid photoelectrochemical cells with photosynthetic reaction centers (RC) has been achieved by using a DNA-based biomimetic antenna. Synthetic dyes Cy3 and Cy5 were chosen and strategically placed in the anntena in such a way that they can collect additional light energy in the visible region of the solar spectrum and transfer it to RC through Förster resonance energy transfer (FRET). The antenna, a DNA templated multiple dye system, is attached to each Rhodobacter sphaeroides RC near the primary donor, P, to facilitate the energy transfer process.

Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial Reaction Centers.

In purple bacterial reaction centers, triplet excitation energy transfer occurs from the primary donor P, a bacteriochlorophyll dimer, to a neighboring carotenoid to prevent photodamage from the generation of reactive oxygen species. The B bacteriochlorophyll molecule that lies between P and the carotenoid on the inactive electron transfer branch is involved in triplet energy transfer between P and the carotenoid. To expand the high-resolution spectral and kinetic information available for describing the mechanism, we investigated the triplet excited state formation and energy transfer pathways in the reaction center of Rhodobacter sphaeroides using pump-probe transient absorption spectroscopy over a broad spectral region on the nanosecond to microsecond time scale at both room temperature and at 77 K.

Orange Carotenoid Protein as a Control Element in an Antenna System Based on a DNA Nanostructure.

Taking inspiration from photosynthetic mechanisms in natural systems, we introduced a light-sensitive photo protective quenching element to an artificial light-harvesting antenna model to control the flow of energy as a function of light intensity excitation. The orange carotenoid protein (OCP) is a nonphotochemical quencher in cyanobacteria: under high-light conditions, the protein undergoes a spectral shift, and by binding to the phycobilisome, it absorbs excess light and dissipates it as heat. By the use of DNA as a scaffold, an antenna system made of organic dyes (Cy3 and Cy5) was constructed, and OCP was assembled on it as a modulated quenching element.

Photocurrent Generation by Photosynthetic Purple Bacterial Reaction Centers Interfaced with a Porous Antimony-Doped Tin Oxide (ATO) Electrode.

The ability to exchange energy and information between biological and electronic materials is critical in the development of hybrid electronic systems in biomedicine, environmental sensing, and energy applications. While sensor technology has been extensively developed to collect detailed molecular information, less work has been done on systems that can specifically modulate the chemistry of the environment with temporal and spatial control. The bacterial photosynthetic reaction center represents an ideal photonic component of such a system in that it is capable of modifying local chemistry via light-driven redox reactions with quantitative control over reaction rates and has inherent spectroscopic probes for monitoring function.

Ultrafast Electron Transfer Kinetics in the LM Dimer of Bacterial Photosynthetic Reaction Center from Rhodobacter sphaeroides.

It has become increasingly clear that dynamics plays a major role in the function of many protein systems. One system that has proven particularly facile for studying the effects of dynamics on protein-mediated chemistry is the bacterial photosynthetic reaction center from Rhodobacter sphaeroides. Previous experimental and computational analysis have suggested that the dynamics of the protein matrix surrounding the primary quinone acceptor, QA, may be particularly important in electron transfer involving this cofactor.

A Three-Enzyme Pathway with an Optimised Geometric Arrangement to Facilitate Substrate Transfer.

Cascade reactions drive and regulate a variety of metabolic activities. Efficient coupling of substrate transport between enzymes is important for overall pathway activity and also controls the depletion of intermediate molecules that drive the reaction forward. Here, we assembled a three-enzyme pathway on a series of DNA nanoscaffolds to investigate the dependence of their activities on spatial arrangement.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes.

Unstructured interactions between peptides and proteins: exploring the role of sequence motifs in affinity and specificity.

Unstructured interactions between proteins and other molecules or surfaces are often described as nonspecific, and have received relatively little attention in terms of their role in biology. However, despite their lack of a specific binding structure, these unstructured interactions can in fact be very selective. The lack of a specific structure for these interactions makes them more difficult to study in a chemically meaningful way, but one approach is statistical, i.

Scalable high-density peptide arrays for comprehensive health monitoring.

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies to report on health fluctuations. Recently, we demonstrated that peptide microarrays can do this through antibody signatures (immunosignatures).

Immunosignature system for diagnosis of cancer.

Although the search for disease biomarkers continues, the clinical return has thus far been disappointing. The complexity of the body's response to disease makes it difficult to represent this response with only a few biomarkers, particularly when many are present at low levels. An alternative to the typical reductionist biomarker paradigm is an assay we call an "immunosignature.

Energy transfer properties of Rhodobacter sphaeroides chromatophores during adaptation to low light intensity.

Time-resolved fluorescence spectroscopy was used to explore the pathway and kinetics of energy transfer in photosynthetic membrane vesicles (chromatophores) isolated from Rhodobacter (Rba.) sphaeroides cells harvested 2, 4, 6 or 24 hours after a transition from growth in high to low level illumination. As previously observed, this light intensity transition initiates the remodeling of the photosynthetic apparatus and an increase in the number of light harvesting 2 (LH2) complexes relative to light harvesting 1 (LH1) and reaction center (RC) complexes.