Mark Platt

Array-based evolution of DNA aptamers allows modelling of an explicit sequence-fitness landscape

Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44 131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.

Structural and electrochemical characterisation of Pt and Pd nanoparticles electrodeposited at the liquid/liquid interface

We present the first reported characterisation by x-ray diffraction and high resolution transmission electron microscopy of metals electrodeposited at the bare and templated liquid/liquid interfaces. Additional structural information is also obtained using ion transfer voltammetry as an in situ characterisation tool. In particular, the metallic deposits are shown to consist of aggregates of discrete nanoparticles, predominantly between 3 and 5 nm in diameter. Deposition of platinum at the liquid/liquid interface is reported for the first time, which enables a preliminary comparison to be made between the growth mechanism of this metal and the growth of palladium, previously reported at this interface.

Electrodeposition of palladium nanoparticles at the liquid-liquid interface using porous alumina templates

Abstract Alumina membranes, with mean pore diameters of 100 nm, have been used as templates to control the electrodeposition of palladium. Deposition occurs at the polarised water–organic interface, leading to the formation of nanoparticles. The particles are formed at the mouth of the alumina pores, the locus of their formation being dictated by the position of the organic–water interface. It is shown that the relative position of the liquid phases with respect to the alumina is controlled by the surface wetting properties of the liquids, rather than gravity. This in turn controls the interfacial position and hence the size of the particles deposited. The presence of the alumina membrane prevents agglomeration. Electrochemical and electron microscopy data are presented in support of this proposed deposition mechanism.

Analysis of a complete DNA–protein affinity landscape

Properties of biological fitness landscapes are of interest to a wide sector of the life sciences, from ecology to genetics to synthetic biology. For biomolecular fitness landscapes, the information we currently possess comes primarily from two sources: sparse samples obtained from directed evolution experiments; and more fine-grained but less authentic information from ‘in silico’ models (such as NK-landscapes). Here we present the entire protein-binding profile of all variants of a nucleic acid oligomer 10 bases in length, which we have obtained experimentally by a series of highly parallel on-chip assays. The resulting complete landscape of sequence-binding pairs, comprising more than one million binding measurements in duplicate, has been analysed statistically using a number of metrics commonly applied to synthetic landscapes. These metrics show that the landscape is rugged, with many local optima, and that this arises from a combination of experimental variation and the natural structural properties of the oligonucleotides.

Aptamer evolution for array-based diagnostics

Closed loop aptameric directed evolution, (CLADE) is a technique enabling simultaneous discovery, evolution, and optimization of aptamers. It was previously demonstrated using a fluorescent protein, and here we extend its applicability with the generation of surface-bound aptamers for targets containing no natural fluorescence. Starting from a random population, in four generations CLADE produced a new aptamer to thrombin with high specificity and affinity. The best aptameric sequence was void of the set of four guanine repeats typifying thrombin aptamers and, thus, highlights the benefits of evolution performed in an environment closely mimicking the final diagnostic application.

Resistive pulse sensing of magnetic beads and supraparticle structures using tunable pores

Tunable pores (TPs) have been used for resistive pulse sensing of 1 μm superparamagnetic beads, both dispersed and within a magnetic field. Upon application of this field, magnetic supraparticle structures (SPSs) were observed. Onset of aggregation was most effectively indicated by an increase in the mean event magnitude, with data collected using an automated thresholding method. Simulations enabled discrimination between resistive pulses caused by dimers and individual particles. Distinct but time-correlated peaks were often observed, suggesting that SPSs became separated in pressure-driven flow focused at the pore constriction. The distinct properties of magnetophoretic and pressure-driven transport mechanisms can explain variations in the event rate when particles move through an asymmetric pore in either direction, with or without a magnetic field applied. Use of TPs for resistive pulse sensing holds potential for efficient, versatile analysis and measurement of nano- and microparticles, while magnetic beads and particle aggregation play important roles in many prospective biosensing applications.

Analysis of aptamer sequence activity relationships

DNA sequences that can bind selectively and specifically to target molecules are known as aptamers. Normally such binding analyses are performed using soluble aptamers. However, there is much to be gained by using an on-chip or microarray format, where a large number of aptameric DNA sequences can be interrogated simultaneously. To calibrate the system, known thrombin binding aptamers (TBAs) have been mutated systematically, producing large populations that allow exploration of key structural aspects of the overall binding motif. The ability to discriminate between background noise and low affinity binding aptamers can be problematic on arrays, and we use the mutated sequences to establish appropriate experimental conditions and their limitations for two commonly used fluorescence-based detection methods. Having optimized experimental conditions, high-density oligonucleotide microarrays were used to explore the entire loop-sequence-functionality relationship creating a detailed model based on over 40 000 analyses, describing key features for quadruplex-forming sequences.

Monitoring aptamer-protein interactions using tunable resistive pulse sensing.

Aptamers are short single-stranded pieces of DNA or RNA capable of binding to analytes with specificity and high affinity. Due to their comparable selectivity, stability, and cost, over the last two decades, aptamers have started to challenge antibodies in their use on many technology platforms. The binding event often leads to changes in the aptamers secondary and tertiary structure; monitoring such changes has led to the creation of many new analytical sensors. Here, we demonstrate the use of a tunable resistive pulse sensing (TRPS) technology to monitor the interaction between several DNA aptamers and their target, thrombin. We immobilized the aptamers onto the surface of superparamagnetic beads, prior to their incubation with the thrombin protein. The protein binding to the aptamer caused a conformational change resulting in the shielding of the polyanion backbone; this was monitored by a change in the translocation time and pulse frequency of the particles traversing the pore. This signal was sensitive enough to allow the tagless detection of thrombin down to nanomolar levels. We further demonstrate the power of TRPS by performing real time detection and characterization of the aptamer-target interaction and measuring the association rates of the thrombin protein to the aptamer sequences.

M13 Bacteriophage‐Activated Superparamagnetic Beads for Affinity Separation

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage.

Particle-by-Particle Charge Analysis of DNA-Modified Nanoparticles Using Tunable Resistive Pulse Sensing

Resistive pulse sensors, RPS, are allowing the transport mechanism of molecules, proteins and even nanoparticles to be characterized as they traverse pores. Previous work using RPS has shown that the size, concentration and zeta potential of the analyte can be measured. Here we use tunable resistive pulse sensing (TRPS) which utilizes a tunable pore to monitor the translocation times of nanoparticles with DNA modified surfaces. We start by demonstrating that the translocation times of particles can be used to infer the zeta potential of known standards and then apply the method to measure the change in zeta potential of DNA modified particles. By measuring the translocation times of DNA modified nanoparticles as a function of packing density, length, structure, and hybridization time, we observe a clear difference in zeta potential using both mean values and population distributions as a function of the DNA structure. We demonstrate the ability to resolve the signals for ssDNA, dsDNA, small changes in base length for nucleotides between 15 and 40 bases long, and even the discrimination between partial and fully complementary target sequences. Such a method has potential and applications in sensors for the monitoring of nanoparticles in both medical and environmental samples.