The HIV-1 frameshift-stimulating (FSS) RNA, a regulatory RNA of critical importance in the virus’ life cycle, has been posited as a novel target for anti-HIV drug development. We report the synthesis and evaluation of triazole-containing compounds able to bind the FSS with high affinity and selectivity. Readily accessible synthetically, these compounds are less toxic than previously reported olefin congeners. We show for the first time that FSS-targeting compounds have antiviral activity against replication-competent HIV in human cells, including a highly cytopathic, multidrug-resistant strain. These results support the viability of the HIV-1 FSS RNA as a therapeutic target and more generally highlight opportunities for synthetic molecule-mediated interference with protein recoding in a wide range of organisms.

Label-free biodetection schemes compatible with standard CMOS fabrication methods constitute an important goal, as these are enabling tools for the mass production of high-sensitivity biosensors. Two-dimensional slab photonic crystal (2D slab-PhC) sensors have been posited as ultrahigh-sensitivity detection components, but to date recognition-mediated detection of viruses or simulants under flow has not been demonstrated. We report the design and optimization of a new W1 waveguide-coupled 2D slab-PhC sensor, with a geometry well suited to virus detection. Proof of concept experiments with fluorescent latex particles verified that the sensor could respond to infiltration of a single particle, both in air and under an aqueous cover layer. Subsequent experiments with antibody-functionalized sensors and virus simulants confirmed the ability of the device to detect virus-sized particles under flow via a recognition-mediated process. This work sets the stage for incorporation of 2D slab-PhC sensors into fully integrated photonic sensor systems.

Human Immunodeficiency Virus (HIV) type 1 uses a −1 programmed ribosomal frameshift (−1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5–10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.

Dynamic Combinatorial Chemistry (DCC) has proven to be a reliable method for identifying hit compounds for target nucleic acid (DNA and RNA) sequences. Typically, these hit compounds are subjected to a lengthy process of optimization via traditional medicinal chemistry. Here, we examine the potential of DCC to also generate and test variations on a hit compound as a method for probing the binding site of an RNA-targeted compound. Specifically, we demonstrate that addition of linker dithiols to a disulfide library containing a known binder to the HIV-1 frameshift-stimulatory RNA (a critical regulator of the HIV life cycle) can yield a mixture of new bridged structures incorporating the dithiol, depending on dithiol structure. Equilibration of this library with the HIV FSS RNA resulted in selection of the original disulfide in preference to bridged structures, suggesting incorporation of the bridge is not compatible with this particular binding site. Application of this strategy to other RNA targets should allow for rapidly profiling the affinity of modified compounds.

Probe molecule immobilization onto surfaces is a critical step in the production of many analytical devices, including labeled and label-free microarrays. New methods to increase the density and uniformity of probe deposition have the potential to significantly enhance the ultimate limits of detection and reproducibility. Hydrogel-based materials have been employed in the past to provide a 3D protein-friendly surface for deposition of antibodies and nucleic acids. However, these methods are susceptible to variation during polymerization of the hydrogel scaffold and provide limited opportunities for tuning deposition parameters on an antibody-by-antibody basis. In this work, a versatile hydrogel nanoparticle deposition method was developed for the production of label-free microarrays and tested in the context of antibody–antigen binding. Poly(N-isopropylacrylamide) nanoparticles (PNIPAM) were conjugated to antibodies using an avidin/biotin system and deposited onto surfaces using a noncontact printing system. After drying, these gel spots formed uniform and thin layers <10 nm in height. The conjugates were characterized with dynamic light scattering, scanning electron microscopy, and atomic force microscopy. We tested this format in the context of tumor necrosis factor-alpha (TNF-α) detection via arrayed imaging reflectometry (AIR), a label-free protein microarray method. This method of probe molecule deposition should be generally useful in the production of microarrays for label-free detection.

Photonic crystals – optical devices able to respond to changes in the refractive index of a small volume of space – are an emerging class of label-free chemical- and bio-sensors. This review focuses on one class of photonic crystal, in which light is confined to a patterned planar material layer of sub-wavelength thickness. These devices are small (on the order of tens to hundreds of microns square), suitable for incorporation into lab-on-a-chip systems, and in theory can provide exceptional sensitivity. We introduce the defining characteristics and basic operation of two-dimensional photonic crystal sensors, describe variations of their basic design geometry, and summarize reported detection results from chemical and biological sensing experiments.

The life cycle of the human immunodeficiency virus type 1 (HIV-1) has an absolute requirement for ribosomal frameshifting during protein translation in order to produce the polyprotein precursor of the viral enzymes. While an RNA stem-loop structure (the “HIV-1 Frameshift Stimulating Signal”, or HIV-1 FSS) controls the frameshift efficiency and has been hypothesized as an attractive therapeutic target, developing compounds that selectively bind this RNA and interfere with HIV-1 replication has proven challenging. Building on our prior discovery of a “hit” molecule able to bind this stem-loop, we now report the development of compounds displaying high affinity for the HIV-1 FSS. These compounds are able to enhance frameshifting more than 50% in a dual-luciferase assay in human embryonic kidney cells, and they strongly inhibit the infectivity of pseudotyped HIV-1 virions.

The near-ubiquity of the involvement of RNA in crucial biological processes is accepted. It is important, therefore, to study and understand the biophysical principles that regulate the function of RNA and its interactions with other molecules (e.g., proteins and antibiotics). Methods enabling the high-throughput determination of RNA–protein binding kinetics and thermodynamics would greatly accelerate understanding of these interactions. To that end, we describe the development of a real-time biomolecular interaction analysis platform based on arrayed imaging reflectometry (AIR) for multiplex analysis of RNA–protein interactions. We demonstrate the use of aqueous AIR by measuring the binding kinetics between muscleblind-like 1 (MBNL1), a splicing regulator protein that plays a pivotal role in the Myotonic Dystrophies and Huntington’s Disease, and several of its RNA targets simultaneously on a microarrayed chip. Using this approach, we observe that the kinetics of MBNL1 binding isolated CUG and repeat CUG RNA sequences (as models for “normal” and “pathogenic” RNA, respectively) are different even though their steady state binding constants are similar. The ability to compare binding kinetics between RNA sequences rapidly and easily may provide insight into the molecular basis of MBNL1-RNA binding, and more generally suggests that AIR can be a powerful tool to enable the label-free, real-time analysis of biomolecular interactions in a high-throughput format.

Nanoscale biosensors have remarkable theoretical sensitivities but often suffer from suboptimal limits of detection in practice. This is in part because the sensing area of nanoscale sensors is orders of magnitude smaller than the total device substrate. Current strategies to immobilize probes (capture molecules) functionalize both sensing and nonsensing regions, leading to target depletion and diminished limits of detection. The difference in topography between these regions on nanoscale biosensors offers a way to selectively address only the sensing area. We developed a bottom-up, topographically selective approach employing self-assembled poly(N-isopropylacrylamide) (PNIPAM) hydrogel nanoparticles as a mask to preferentially bind target to only the active sensing region of a photonic crystal (PhC) biosensor. This led to over an order of magnitude improvement in the limit of detection for the device, in agreement with finite element simulations. Since the sensing elements in many nanoscale sensors are topographically distinct, this approach should be widely applicable.

•Various solution-phase aminosilane deposition methods were compared to vapor-phase films.•Films of APTES, APDMES and APMDES were prepared from solution and vapor-phase.•Silane film quality was assessed by thickness, hydrophilicity and surface roughness.•Two solution-phase methods produced silane films comparable to vapor-deposited films.The uniformity of aminosilane layers typically used for the modification of hydroxyl bearing surfaces such as silicon dioxide is critical for a wide variety of applications, including biosensors. However, in spite of many studies that have been undertaken on surface silanization, there remains a paucity of easy-to-implement deposition methods reproducibly yielding smooth aminosilane monolayers. In this study, solution- and vapor-phase deposition methods for three aminoalkoxysilanes differing in the number of reactive groups (3-aminopropyl triethoxysilane (APTES), 3-aminopropyl methyl diethoxysilane (APMDES) and 3-aminopropyl dimethyl ethoxysilane (APDMES)) were assessed with the aim of identifying methods that yield highly uniform and reproducible silane layers that are resistant to minor procedural variations. Silane film quality was characterized based on measured thickness, hydrophilicity and surface roughness. Additionally, hydrolytic stability of the films was assessed via these thickness and contact angle values following desorption in water. We found that two simple solution-phase methods, an aqueous deposition of APTES and a toluene based deposition of APDMES, yielded high quality silane layers that exhibit comparable characteristics to those deposited via vapor-phase methods.

Rapid, sensitive, and selective detection of viruses is critical for applications in medical diagnostics, biosecurity, and environmental safety. In this article, we report the application of a point-defect-coupled W1 photonic crystal (PhC) waveguide biosensor to label-free optical detection of viruses. Fabricated on a silicon-on-insulator (SOI) substrate using electron-beam (e-beam) lithography and reactive-ion-etching, the PhC sensing platform allows optical detection based on resonant mode shifts in response to ambient refractive index changes produced by infiltration of target biomaterial within the holes of the PhC structure. Finite difference time domain (FDTD) calculations were performed to assist with design of the sensor, and to serve as a theoretical benchmark against which experimental results could be compared. Using Human Papillomavirus virus-like particles (VLPs) spiked in 10% fetal bovine serum as a model system, we observed a limit of detection of 1.5 nM in simple (buffer only) or complex (10% serum) sample matrices. The use of anti-VLP antibodies specific for intact VLPs with the PhC sensors provided highly selective VLP detection.Highlights► 2-D Photonic Crystals are optical sensors with an exceptionally small sensing volume. ► We conducted FDTD calculations that suggest single virus detection is possible. ► Detection of human papillomavirus virus-like particles (VLPs) was demonstrated. ► High selectivity was obtained in buffer and in serum.

One of the critical steps in the development of an analytical technique is to confirm that its experimental response correlates with predictions derived from the theoretical framework on which it is based. This validates the technique quantitatively and, in the case of a biosensor, facilitates a correlation of the sensor’s output signal to the concentration of the analyte being tested. Herein we report studies demonstrating that the quantitative response of arrayed imaging reflectometry (AIR), a highly sensitive label-free biosensing method, is a predictable function of the probe and analyte properties. We first incorporated a standard one-site Langmuir binding model describing probe–analyte interactions at the surface into the theoretical model for thickness-dependent reflectance in AIR. This established a hypothetical correlation between the analyte concentration and the AIR response. Spectroscopic ellipsometry, surface plasmon resonance, and AIR were then used to validate this model for two biomedically important proteins, fibroblast growth factor-2 and vascular endothelial growth factor. While our studies demonstrated that the 1:1 one-site Langmuir model accurately described the observed response of macrospot AIR arrays, either a two-site Langmuir model or a Sips isotherm better described the behavior of AIR microarrays. These studies confirmed the quantitative performance of AIR across a range of probe–analyte affinities. Furthermore, the methodology developed here can be extended to other label-free biosensing platforms, thus facilitating a more accurate and quantitative interpretation of the sensor response.

The emerging field of plasmonics, the study of electromagnetic responses of metal nanostructures, has revealed many novel signal enhancing phenomena. As applied to the development of label-free optical DNA biosensors, it is now well established that plasmon-based surface enhanced spectroscopies on nanostructured metal surfaces or metal nanoparticles can markedly improve the sensitivity of optical biosensors, with some showing great promise for single molecule detection. In this review, we first summarize the basic concepts of plasmonics in metal nanostructures, as well as the characteristic optical phenomena to which plasmons give rise. We will then describe recent advances in optical DNA biosensing systems enabled by metal nanoparticle-derived plasmonic effects, including the use of surface enhanced Raman scattering (SERS), colorimetric methods, “scanometric” processes, and metal-enhanced fluorescence (MEF).

Production of the Gag-Pol polyprotein in human immunodeficiency virus (HIV) requires a −1 ribosomal frameshift, which is directed by a highly conserved RNA stem−loop. Building on our discovery of a set of disulfide-containing peptides that bind this RNA, we describe medicinal chemistry efforts designed to begin to understand the structure−activity relationships and RNA sequence−selectivity relationships associated with these compounds. Additionally, we have prepared analogues incorporating an olefin or saturated hydrocarbon bioisostere of the disulfide moiety, as a first step toward enhancing biostability. The olefin-containing compounds exhibit affinity comparable to the lead disulfide and, importantly, have no discernible toxicity when incubated with human fibroblasts at concentrations up to 1 mM.

Localized surface plasmons of metallic nanoparticles can strongly amplify the magnitude of the surrounding electric field. This in turn enhances fluorescence from nearby fluorophores. However, little is known regarding how time-dependent changes in nanoparticle structure due to exposure to the ambient environment affect their behavior in plasmonic devices. Here, we report the interesting finding that the aging of a nanostructured Ag substrate in ambient atmosphere markedly improves the fluorescence signal of a plasmonic-based DNA detection system. The effect can be observed with an exposure time as short as two days, and a nearly 17-fold signal enhancement can be achieved with 30 days of aging. Analysis of substrate surface topography by atomic force microscopy (AFM) reveals a substantial change in nanoparticle morphology as the substrates age despite being covalently attached to a solid dry substrate. Nanoparticle morphological changes also manifest in extinction spectra. This process can be further accelerated by light. Together, our findings address the important question of Ag nanoparticle stability over time and its potential ramifications for plasmon-enabled sensors. They also imply that nanoparticle aging may be used strategically to tune nanoparticle size and geometry and plasmon spectrum, which may be beneficial for studies on plasmonics as well as sensor optimization.

The dramatic local electric-field enhancement property of Ag nanoparticles was used as the basis to significantly increase the signal output of a novel label-free (or “self-labeled”) fluorescence-based DNA detection system. In response to identical amounts of analyte, nanostructured Ag substrates provided a posthybridization fluorescent sensor response over 10-fold larger than the response from planar Au substrates. Detection performance strongly depended upon the Ag substrate roughness. Consistent with work by others on metal-enhanced fluorescence, fluorescence intensity also depended strongly on the distance between the fluorophore and the Ag substrate surface. Adjusting the surface roughness, amount of the Ag deposited on the surface, and the DNA probe length allowed for production of an optimized response. In addition to constituting a novel label-free DNA sensor, we anticipate that these structures will provide a unique platform for testing concepts in plasmonics.Keywords: Ag nanoparticles; DNA detection; DNA hairpins; metal enhanced fluorescence

Uniform spot morphology is of critical importance in the fabrication and successful use of protein arrays, and solution additives are often needed to ensure good spot quality. Whereas hydroxyl-bearing molecules such as glycerol have found wide use, in our experience these reduce the efficiency of probe immobilization (particularly in the context of aldehyde-terminated surfaces). Here, we report a series of non-nucleophilic molecules that can be used as additives to improve spot homogeneity in protein arrays. Arrayed imaging reflectometry, a label-free optical biosensing technique, has been used along with spectroscopic ellipsometry to test the spot homogeneity, antibody immobilization efficiency, and activity of antihuman IgG arrays prepared with these non-nucleophilic additives on glutaraldehyde surfaces. It has been determined that 0.1% v/v 12-crown-4 performs optimally in MPBS buffer.

Hairpin DNA sequences are widely used as probes for oligonucleotides in a broad range of assays, often as “molecular beacons”. A potential disadvantage of the standard methodology for molecular beacon design is the need to add several self-complementary bases to each end of the probe, since these do not correspond to the target sequence. We describe a conceptually new method of hairpin DNA probe identification, in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within an expressed gene having the requisite hairpin structure. Intuitively, such probes should have significantly improved performance over “traditional” hairpin probes, because they are fully complementary with the target. We present experimental evidence verifying this hypothesis for a series of hairpin probes targeting the pag gene of Bacillus anthracis.

The ability to carry out simultaneous orthogonal exchange chemistries has opened new opportunities for increasing the numerical and structural diversity accessible to Dynamic Combinatorial Chemistry. We present proof-of-concept experiments demonstrating this concept is transferrable to resin-bound DCC, facilitating the generation and analysis of libraries with greater structural diversity.