RNA Polymerase Ribozyme That Recognizes the Template-Primer Complex through Tertiary Interactions.

RNA enzymes (ribozymes) often rely on specific base-pairing interactions to engage RNA substrates, which limits the substrate sequence generality of these enzymes. An RNA polymerase ribozyme that was previously optimized by directed evolution to operate in a more efficient and sequence-general manner can now recognize the RNA template, RNA primer, and incoming nucleoside 5'-triphosphate (NTP) entirely through tertiary interactions. As with proteinaceous polymerases, these tertiary interactions are largely agnostic to the sequence of the template, which is an essential property for the unconstrained transmission of genetic information. The polymerase ribozyme exhibits Michaelis-Menten saturation kinetics, with a catalytic rate of 0.1-1 min-1 and a Km of 0.1-1 µM. Earlier forms of the polymerase did not exhibit a saturable substrate binding site, but this property emerged over the course of directed evolution as the ribozyme underwent a structural rearrangement of its catalytic center. The optimized polymerase makes tertiary contacts with both the template and primer, including a critical interaction at the C2' position of the template nucleotide that opposes the 3'-terminal nucleotide of the primer. UV cross-linking studies paint a picture of how several portions of the ribozyme, including regions that were remodeled by directed evolution, come together to position the template, primer, and NTP within the active site for RNA polymerization.

Aptamer-Mediated Reversible Transactivation of Gene Expression by Light.

The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.

A blue light receptor that mediates RNA binding and translational regulation.

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL-RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities.

Advances in developing rapid, reliable and portable detection systems for alcohol.

Development of portable, reliable, sensitive, simple, and inexpensive detection system for alcohol has been an instinctive demand not only in traditional brewing, pharmaceutical, food and clinical industries but also in rapidly growing alcohol based fuel industries. Highly sensitive, selective, and reliable alcohol detections are currently amenable typically through the sophisticated instrument based analyses confined mostly to the state-of-art analytical laboratory facilities. With the growing demand of rapid and reliable alcohol detection systems, an all-round attempt has been made over the past decade encompassing various disciplines from basic and engineering sciences. Of late, the research for developing small-scale portable alcohol detection system has been accelerated with the advent of emerging miniaturization techniques, advanced materials and sensing platforms such as lab-on-chip, lab-on-CD, lab-on-paper etc. With these new inter-disciplinary approaches along with the support from the parallel knowledge growth on rapid detection systems being pursued for various targets, the progress on translating the proof-of-concepts to commercially viable and environment friendly portable alcohol detection systems is gaining pace. Here, we summarize the progress made over the years on the alcohol detection systems, with a focus on recent advancement towards developing portable, simple and efficient alcohol sensors.

Multifaceted analyses of the interactions between human heart type fatty acid binding protein and its specific aptamers.

BACKGROUND: Aptamer-protein interaction studies have been mainly confined to dissociation constant (Kd) determination. A combinatorial approach involving limited proteolysis mass spectroscopy, molecular docking and CD studies is reported here to elucidate the specific interactions involved. METHODS: To generate aptamers specific for human FABP3, SELEX was performed incorporating counter SELEX cycles against control FABPs and GST tag, followed by their characterization by EMSA, CD and SVD analysis. Based on computationally obtained aptamer-protein complex models, the interacting aptamer, and protein residues were predicted and supported by limited proteolysis experiments. RESULTS: Two aptamers N13 and N53 specific for human fatty acid binding protein (FABP3) were isolated with corresponding Kd of 0.0743±0.0142µM and 0.3337±0.1485µM for FABP3 interactions. Both aptamers possess stable B-DNA structures at salt concentration of 100mM and pH range (6-9). The N13 aptamer led interaction involved 3 salt bridges and 2 hydrogen bonds, whereas N53 had 2 salt bridges with 8 hydrogen and 7 hydrophobic interactions. CONCLUSIONS: The aptamers generated are the first to be reported against human FABP3. The higher interaction footprint of N53 incited synergistic conformational changes in both N53 and FABP3 during interaction, leading to a decline in binding affinity in comparison to N13 which corroborated to the calculated Kd values. GENERAL SIGNIFICANCE: This combinatorial method may be used to retrieve the possible specific binding modes and interaction patterns involved in large aptamer-protein complexes. Thus the method can be exploited to identify the optimum aptamer length for in-depth structure-function studies and its tailored applications.

A low cost design and fabrication method for developing a leak proof paper based microfluidic device with customized test zone.

This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 µl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 µl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones.

Selective and sensitive detection of free bilirubin in blood serum using human serum albumin stabilized gold nanoclusters as fluorometric and colorimetric probe.

We report here a fluorescence quenching based non-enzymatic method for sensitive and reliable detection of free bilirubin in blood serum samples using human serum albumin (HSA) stabilized gold nanoclusters (HSA-AuNCs) as fluorescent probe. The fluorescence of the nanoclusters was strongly quenched by bilirubin in a concentration dependent manner by virtue of the inherent specific interaction between bilirubin and HSA. A strong binding constant of 0.55×10(6) L mole(-1) between the HSA-AuNC and bilirubin was discerned. The nano clusters each with size ~1.0 nm (in diameter) and a core of Au18 were homogeneously distributed in HSA molecules as revealed from the respective high resolution transmission electron microscopic and mass spectroscopic studies. The fluorescence quenching phenomena which obeyed a simple static quenching mechanism, was utilized for interference free detection of bilirubin with minimum detection limit (DL) of 248±12 nM (S/N=3). The fluorescence response of HSA-AuNCs against bilirubin was practically unaltered over a wide pH (6-9) and temperature (25-50 °C) range. Additionally, peroxidase-like catalytic activity of these nanoclusters was exploited for colorimetric detection of bilirubin in serum sample with a DL of 200±19 nM by following the decrease in absorbance (at λ440 nm) of the reaction and its rate constant (Kp) of 2.57±0.63 mL µg(-1) min(-1). Both these fluorometric and colorimetric methods have been successfully used for detection of free bilirubin in blood serum samples.

A novel amperometric alcohol biosensor developed in a 3rd generation bioelectrode platform using peroxidase coupled ferrocene activated alcohol oxidase as biorecognition system.

Alcohol oxidase (AOx) with a two-fold increase in efficiency (Kcat/Km) was achieved by physical entrapment of the activator ferrocene in the protein matrix through a simple microwave based partial unfolding technique and was used to develop a 3rd generation biosensor for improved detection of alcohol in liquid samples. The ferrocene molecules were stably entrapped in the AOx protein matrix in a molar ratio of ~3:1 through electrostatic interaction with the Trp residues involved in the functional activity of the enzyme as demonstrated by advanced analytical techniques. The sensor was fabricated by immobilizing ferrocene entrapped alcohol oxidase (FcAOx) and sol-gel chitosan film coated horseradish peroxidase (HRP) on a multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode through layer-by-layer technique. The bioelectrode reactions involved the formation of H2O2 by FcAOx biocatalysis of substrate alcohol followed by HRP-catalyzed reduction of the liberated H2O2 through MWCNT supported direct electron transfer mechanism. The amperometric biosensor exhibited a linear response to alcohol in the range of 5.0 × 10(-6) to 30 × 10(-4)mol L(-1) with a detection limit of 2.3 × 10(-6) mol L(-1), and a sensitivity of 150 µA mM(-1) cm(-2). The biosensor response was steady for 28 successive measurements completed in a period of 5h and retained ~90% of the original response even after four weeks when stored at 4 °C. The biosensor was successfully applied for the determination of alcohol in commercial samples and its performance was validated by comparing with the data obtained by GC analyses of the samples.

An overview on alcohol oxidases and their potential applications.

Alcohol oxidases (Alcohol: O2 Oxidoreductase; EC 1.1.3.x) are flavoenzymes that catalyze the oxidation of alcohols to the corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. Based on substrate specificity, alcohol oxidases may be categorized broadly into four different groups namely, (a) short chain alcohol oxidase (SCAO), (b) long chain alcohol oxidase (LCAO), (c) aromatic alcohol oxidase (AAO), and (d) secondary alcohol oxidase (SAO). The sources reported for these enzymes are mostly limited to bacteria, yeast, fungi, plant, insect, and mollusks. However, the quantum of reports for each category of enzymes considerably varies across these sources. The enzymes belonging to SCAO and LCAO are intracellular in nature, whereas AAO and SAO are mostly secreted to the medium. SCAO and LCAO are invariably reported as multimeric proteins with very high holoenzyme molecular masses, but the molecular characteristics of these enzymes are yet to be clearly elucidated. One of the striking features of the alcohol oxidases that make them distinct from the widely known alcohol dehydrogenase is the avidly bound cofactor to the redox center of these enzymes that obviate the need to supplement cofactor during the catalytic reaction. These flavin-based redox enzymes have gained enormous importance in the development of various industrial processes and products primarily for developing biosensors and production of various industrially useful carbonyl compounds. The present review provides an overview on alcohol oxidases from different categories focusing research on these oxidases during the last decade along with their potential industrial applications.

Heart type fatty acid binding protein: structure, function and biosensing applications for early detection of myocardial infarction.

Heart type fatty acid binding protein (HFABP) as an early marker of cardiac injury holds a promising future with studies indicating surpassing performance as compared to myoglobin. As a plasma marker, this cytoplasmic protein owing to its small size (∼15kDa) and water solubility, appears readily in the blood-stream following cardiomyocyte damage, reaching peak levels within 6h of symptom onset. Low plasma levels of HFABP as compared to tissue levels indicate that minute amounts of the protein when released during myocardial infarction leads to a greater proportional rise. These parameters of kinetic release make it an ideal candidate for rapid assessment of acute myocardial infarction (AMI). The need for development of rapid immunoassays and immunotests so as to use HFABP as an early marker for AMI exclusion is tremendous. In the present review, we outline the various immunoassays and immunosensors developed so far for the detection of HFABP in buffer, plasma or whole blood. The principles behind the detection techniques along with their performance parameters compared to standard ELISA techniques are elucidated.