Manish Biyani

cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA–protein fusions

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Solid-phase translation and RNA-protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA.

A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3′ ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution.

Microintaglio Printing of Biomolecules and Its Application to In situ Production of Messenger Ribonucleic Acid Display Microarray

A new molecular printing technology for a simple and robust patterning of biomolecules was developed and applied to producing a genotype-linked protein microarray. A microarray of messenger ribonucleic acids (mRNAs) encoding the green fluorescent protein was patterned by microintaglio printing using a micromold comprising an array of uniformly arranged 5-µm-diameter holes at a density of 10,000 per mm2. Furthermore, one-step conversion from an mRNA microarray into an mRNA-protein fusion microarray was performed by simultaneous cell-free protein synthesis and fusion reaction using a puromycin-labeled oligonucleotide linker. The set of developed technologies provides a powerful means of in vitro protein evolution.

A bulk sub-femtoliter in vitro compartmentalization system using super-fine electrosprays

The extreme miniaturization of biological and chemical assays in aqueous-droplet compartments enables spatiotemporal control for large-scale parallel experimentation and can thus permit new capabilities for “digitizing” directed molecular evolution methodologies. We report a remarkably facile bulk method to generate mega-scale monodisperse sub-femtoliter aqueous droplets by electrospray, using a prototype head with super-fine inkjet technology. Moreover, the electrostatic inkjet nozzle that injects the aqueous phase when immersed within an immiscible phase (an optimized oil/surfactant mixture) has the advantage of generating cell-like sub-femtoliter compartments for biomolecule encapsulation and successive biological and chemical reactions. Sub-femtoliter droplets of both liquid (water-in-oil, volumes ranging from 0.2 to 6.4 fL) and gel bead (agarose-in-oil, volume ranging from 0.3 to 15.6 fL) compartments with average sizes of 1.3 μm and 1.5 μm, respectively, were successfully generated using an inkjet nozzle at a speed of more than 105 droplets per second. We demonstrated the applicability of this system by synthesizing fluorescent proteins using a cell-free expression system inside electrosprayed sub-femtoliter droplets at an accelerated rate, thereby extending the utility of in vitro compartmentalization with improved analytical performance for a top-down artificial cellular system.

Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25–100 µm features.

Detection of ultra-low levels of DNA changes by drinking water: epidemiologically important finding.

The safety of drinking water is essential to our health. In this context, the mutagenicity of water needs to be checked strictly. However, from the methodological limit, the lower concentration (less than parts per million) of mutagenicity could not be detected, though there have been of interest in the effect of less concentration mutagens. Here, we describe a highly sensitive mutation assay that detects mutagens at the ppb level, termed genome profiling-based mutation assay (GPMA). This consists of two steps; (i) Escherichia coli culture in the medium with/without mutagens and (ii) Genome profiling (GP) method (an integrated method of random PCR, temperature gradient gel electrophoresis and computer-aided normalization). Owing to high sensitivity of this method, very low concentration of mutagens in tap water could be directly detected without introducing burdensome concentration processes, enabling rapid measurement of low concentration samples. Less expectedly, all of the tap waters tested (22 samples) were shown to be significantly mutagenic while mineral waters were not. Resultantly, this article informs two facts that the GPMA method is competent to measure the mutagenicity of waters directly and the experimental results supported the former reports that the city tap waters contain very low level of mutagenicity reagent trihalomethanes.

Evaluation of poly(dimethylsiloxane) microreactors for pattern size miniaturization of microintaglio-printing-based protein microarray

We developed a soft lithography-based microintaglio printing method for fabricating robot-free high-density protein microarrays using a poly(dimethylsiloxane) (PDMS) microchamber array. This method provides a simple approach to simultaneously synthesize and capture functional protein microarrays (with 25?100 ?m resolution in this study) directly from DNA microarrays in situ on a chip without a spotter and therefore can be an extremely effective tool for the miniaturization of arrays. However, minimizing the scale, which increases the PDMS surface area to sample volume ratio, can alter arraying outcomes because of interfacial phenomena. In this study, we evaluated the suitability of PDMS for miniaturizing cell-free synthesis-based protein microarrays and showed that the amount of a green fluorescent protein synthesized in situ inside PDMS microchambers monotonically decreased with decreasing microchamber (pattern) size and that the protein could not be detected in microchambers with a diameter smaller than 25 ?m. The impact of absorption and/or adsorption on the PDMS surface on protein synthesis inside the microchamber was observed, which was reduced by coating techniques. The results obtained here clearly suggest that PDMS interfacial phenomena can be suppressed while obtaining the benefits of cell-free protein synthesis and microintaglio printing, as a step toward developing ultrahigh-density protein microarrays.

DEP-On-Go for Simultaneous Sensing of Multiple Heavy Metals Pollutants in Environmental Samples

We describe a simple and affordable “Disposable electrode printed (DEP)-On-Go” sensing platform for the rapid on-site monitoring of trace heavy metal pollutants in environmental samples for early warning by developing a mobile electrochemical device composed of palm-sized potentiostat and disposable unmodified screen-printed electrode chips. We present the analytical performance of our device for the sensitive detection of major heavy metal ions, namely, mercury, cadmium, lead, arsenic, zinc, and copper with detection limits of 1.5, 2.6, 4.0, 5.0, 14.4, and, 15.5 μg·L−1, respectively. Importantly, the utility of this device is extended to detect multiple heavy metals simultaneously with well-defined voltammograms and similar sensitivity. Finally, “DEP-On-Go” was successfully applied to detect heavy metals in real environmental samples from groundwater, tap water, house dust, soil, and industry-processed rice and noodle foods. We evaluated the efficiency of this system with a linear correlation through inductively coupled plasma mass spectrometry, and the results suggested that this system can be reliable for on-site screening purposes. On-field applications using real samples of groundwater for drinking in the northern parts of India support the easy-to-detect, low-cost (<1 USD), rapid (within 5 min), and reliable detection limit (ppb levels) performance of our device for the on-site detection and monitoring of multiple heavy metals in resource-limited settings.

Temperature-controlled microintaglio printing for high-resolution micropatterning of RNA molecules

We have developed an advanced microintaglio printing method for fabricating fine and high-density micropatterns and applied it to the microarraying of RNA molecules. The microintaglio printing of RNA reported here is based on the hybridization of RNA with immobilized complementary DNA probes. The hybridization was controlled by switching the RNA conformation via the temperature, and an RNA microarray with a diameter of 1.5 µm and a density of 40,000 spots/mm(2) with high contrast was successfully fabricated. Specifically, no size effects were observed in the uniformity of patterned signals over a range of microarray feature sizes spanning one order of magnitude. Additionally, we have developed a microintaglio printing method for transcribed RNA microarrays on demand using DNA-immobilized magnetic beads. The beads were arrayed on wells fabricated on a printing mold and the wells were filled with in vitro transcription reagent and sealed with a DNA-immobilized glass substrate. Subsequently, RNA was in situ synthesized using the bead-immobilized DNA as a template and printed onto the substrate via hybridization. Since the microintaglio printing of RNA using DNA-immobilized beads enables the fabrication of a microarray of spots composed of multiple RNA sequences, it will be possible to screen or analyze RNA functions using an RNA microarray fabricated by temperature-controlled microintaglio printing (TC-µIP).

Biomolecular Display Technology: A New Tool for Drug Discovery

Abstract The identification of molecules with desired function is of a great significance in biology and medicine. Display technologies represent a new tool for the drug discovery by facilitating the screening of novel biomolecules against any target of choice. This chapter reviews the development of in vitro display technologies and their application into drug discovery, focusing on challenges and perspective for the rapid and efficient modern drug discovery process.