Rajesh Ahirwar

In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules

Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5′-GGGGUCAAGGUGACCCC-3′) having a binding constant (Ka) of 1.02 ± 0.1 × 108 M−1 as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases.

Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis

Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A.

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (Ka = 1.55±0.298×108 M-1; ΔH = 4.32×104±801.1 cal/mol; ΔS = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.

Screening and identification of a DNA aptamer to concanavalin A and its application in food analysis.

Herein, a novel aptamer that targets concanavalin A (Con A), a plant lectin, is isolated using systematic evolution of ligands by an exponential enrichment (SELEX) technique. Nine rounds of SELEX screening over an agarose spin column have resulted in enrichment of eight sequences having high affinity to Con A. The highest affinity sequence was selected as a potent aptamer and characterized it in detail. The evolved Con A aptamer (Con A-aptabody) is a 41 nt ssDNA that binds the Con A specifically with a dissociation constant of 172.7 ± 29.7 nM. In silico analyses predict the Con A-aptabody to form G-quadruplex due to its G-rich sequence (GGAAGGCGGAGGG). A detection method developed using Con A-aptabody is found to have a detection range of 10-750 ng/mL with limits of detection and quantification being 13.22 and 44.09 ng/mL, respectively. The utility of the method is demonstrated by analyzing jack bean (Canavalia ensiformis), kidney bean (Phaseolus vulgaris), wheat (Triticum spp.), mung bean (Vigna radiata), and lentil (Lens culinaris) for their Con A contents. Hence, the developed Con A-aptabody provides a useful substitute to Con A-antibody for food analysis and related applications.

Microwave non-thermal effect reduces ELISA timing to less than 5 minutes

This report demonstrates that microwave-mediated ELISA, which occurs in less than 5 minutes, is due to a microwave non-thermal or a microwave-specific effect rather than the microwave heating effect. To decipher the non-thermal effect, we have designed a system that mimics the time-dependent temperature rise of a reaction mixture or buffer in the wells of a polystyrene microtiter plate similar to that of microwave exposure. This system is used as an alternative to the microwave thermal effect (microwave-thermal-alternate or MTA). We have carried out ELISA for the detection of human IgG in a time-dependent manner under microwave irradiation in a microwave oven, and by thermal incubation by a specially designed MTA. ELISA results carried out by microwave exposure in 4 min 40 s are akin to 18 h conventional ELISA, whereas no significant ELISA values are obtained by microwave-thermal-alternate, illustrating the predominance of the microwave non-thermal effect over the microwave thermal effect in microwave-mediated ELISA. We postulate that the microwave specific effect is a microwave catalytic effect acting by lowering the activation energy of reactants.

Microwave-mediated enzymatic modifications of DNA

Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA.

Exploring the flexible chemistry of 4-fluoro-3-nitrophenyl azide for biomolecule immobilization and bioconjugation

AbstractBioconjugation and functionalization of polymer surfaces are two major tasks in materials chemistry which are accomplished using a variety of coupling agents. Immobilization of biomolecules onto polymer surfaces and the construction of bioconjugates are essential requirements of many biochemical assays and chemical syntheses. Different linkers with a variety of functional groups are used for these purposes. Among them, the benzophenones, aryldiazirines, and arylazides represent the most commonly used photolinker to produce the desired chemical linkage upon their photo-irradiation. In this review, we describe the versatile applications of 4-fluoro-3-nitrophenyl azide, one of the oldest photolinkers used for photoaffinity labeling in the late 1960s. Surprisingly, this photolinker, historically known as 1-fluoro-2-nitro-4-azidobenzene (FNAB), has remained unexplored for a long time because of apprehension that FNAB forms ring-expanded dehydroazepine as a major product and hence cannot activate an inert polymer. The first evidence of photochemical activation of an inert surface by FNAB through nitrene insertion reaction was reported in 2001, and the FNAB-activated surface was found to conjugate a biomolecule without any catalyst, reagent, or modification. FNAB has distinct advantages over perfluorophenyl azide derivatives, which are contemporary nitrene-generating photolinkers, because of its simple, single-step preparation and ease of thermochemical and photochemical reactions with versatile polymers and biomolecules. Covering these aspects, the present review highlights the flexible chemistry of FNAB and its applications in the field of surface engineering, immobilization of biomolecules such as antibodies, enzymes, cells, carbohydrates, oligonucleotides, and DNA aptamers, and rapid diagnostics. Graphical AbstractAn overview of the FNAB-engineered activated polymer surfaces for covalent ligation of versatile biomolecules

BSA blocking in enzyme-linked immunosorbent assays is a non-mandatory step: a perspective study on mechanism of BSA blocking in common ELISA protocols

BSA blocking is a routine practice among clinicians and researchers working on immunoassays throughout the world. The primary role of BSA is to prevent the non-specific binding by blocking the leftover spaces over solid surface after immobilization of a capture biomolecule. However, the acquired diversity of BSA blocking has remained conflicted on nature of the solid surfaces used, antigen–antibody combinations, and their concentrations. Here, we investigate the necessity of BSA blocking in common ELISA protocols by performing ELISA detection of human-IgG, rabbit-IgG, human-IgE, concanavalin A and hepatitis C core antigen with and without BSA blocking on different microplates and with different concentrations of analytes. We found that irrespective of solid surfaces or analyte concentrations, the ELISA protocols with and without-BSA blocking produce similar outcomes when performed with PBST washing. However, if PBS instead of PBST is used for washing in assays with BSA blocking, the chances of wrong predictions enhance significantly. Further, by using FITC-tagged BSA, we have found that BSA binds weakly to microplate surface and escapes during PBST washing. Again, if PBS rather than PBST is used in combination to BSA blocking, case-dependent non-specificity is added to ELISA results. Based on these observations, we suggest to empirically determine the absolute necessity of BSA-blocking, as majority of ELISA protocols do not need BSA-blocking.

Microwave-Assisted Rapid Enzymatic Synthesis of Nucleic Acids

ABSTRACT Herein we report microwave-induced enhancement of the reactions catalyzed by Escherichia coli DNA polymerase I and avian myeloblastosis virus-reverse transcriptase. The reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10–50 seconds. In contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. Each of the reactions was carried out for different duration of microwave exposure time to find the optimum reaction time. The products produced by the respective enzyme upon microwave irradiation of the reaction mixtures were identical to that produced by the conventional procedures. As the microwave-assisted reactions are rapid, microwave could be a useful alternative to the conventional and time consuming procedures of enzymatic synthesis of nucleic acids.

A Simple and Efficient Method for Removal of Phenolic Contaminants in Wastewater Using Covalent Immobilized Horseradish Peroxidase

In the light of the increasing burden of pollutants in major rivers and stringent environmental legislation, adaptation to eco-friendly treatment approaches is desperately required to decontaminate industrial effluents before its discharge to rivers and other fresh water-bodies. In present study, we have designed a simple and efficient method for removal of phenols from effluent wastewater using an immobilized preparation of HRP (horseradish peroxidase). The enzyme was isolated in bulk amount from the roots of the Armoracia rusticana and covalently immobilized to polycarbonate supports using a photolabile linker FNAB (1-fluoro-2-notro-4-azidobenzene). The immobilized enzyme showed enhanced storage stability and reusability. The immobilized HRP was subsequently used for degradation of phenols in sewage and spiked wastewater. The phenol content of spiked wastewater was reduced to 93% in the 3 L reactor following treatment with immobilized HRP and H2O2. The improvement in the quality of water upon treatment was reflected by the changes in pH, conductivity, TDS (total dissolved salts) and biodegradation of organic contents as indicated by 77% and 87% reduction in COD (chemical oxygen demand) and BOD (biochemical oxygen demand) respectively in the analyzed sample.