Showing papers by "Andrew D. Ellington published in 2010"
TL;DR: A series of aptamers currently in development may change how nucleic acid therapeutics are perceived and will increasingly find use in concert with other therapeutic molecules and modalities.
Abstract: Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.
1,707 citations
TL;DR: It is found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between ∼1% and >10% of the total repertoire.
Abstract: Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.
338 citations
TL;DR: A fully integrated biosensor 10 × 10 array in a standard complementary metal-oxide semiconducor process, which takes advantage of electrochemical impedance spectroscopy (EIS), which is able to detect various biological analytes in real time and without the need for molecular labels.
Abstract: In this paper, we present a fully integrated biosensor 10 × 10 array in a standard complementary metal-oxide semiconducor process, which takes advantage of electrochemical impedance spectroscopy (EIS). We also show that this system is able to detect various biological analytes, such as DNA and proteins, in real time and without the need for molecular labels. In each pixel of this array, we implement a biocompatible Au electrode transducer and embedded sensor circuitry which takes advantage of the coherent detector to measure the impedance of the associated electrode-electrolyte interface. This chip is capable of concurrently measuring admittance values as small as 10-8 Ω-1 within the array with the detection dynamic range of more than 90 dB in the frequency range of 10 Hz-50 MHz.
242 citations
TL;DR: A versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters is described, providing key insights into clinically relevant phenotypes in low-cell-number/high- density bacterial populations.
Abstract: Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]) Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 10(8) bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells Indeed, bacterial clusters containing 10(1) to 10(5) cells are important for transmission of many bacterial pathogens Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial "lobster traps") defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules Within these traps, bacteria divide normally into extremely dense (10(12) cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations
147 citations
TL;DR: The findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.
Abstract: Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.
129 citations
TL;DR: A newly selected anti-receptor (anti-EGFR) aptamer was conjugated to gold nanoparticles via a facile hybridization method and was found to specifically and quantitatively direct the delivery of gold nanoparticle to cells expressing EGFR through receptor-mediated endocytosis.
119 citations
TL;DR: A robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method and a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy is presented.
Abstract: We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.
102 citations
18 Mar 2010
TL;DR: The main design challenge of biosensors is to increase the SNR and DR while minimizing the complexity of both the assay and the detector.
Abstract: Biosensors are one of the fundamental detection platforms in biotechnology. They take advantage of unique biomolecular interactions to capture and detect specific analytes on a surface. The detection versatility of biosensors has always been their key advantage and it has been demonstrated that they can detect almost any analyte such as DNA, proteins, metabolites, and even micro-organisms. However, the achievable SNR and detection DR of biosensors can be very low. This is due to the fact that the capturing processes in biosensors suffer from a significant amount of biological interference (i.e., non-specific bindings) and biochemical noise which typically necessitate the use of complex biochemical labeling processes and sophisticated detectors [1]. Hence, the main design challenge of biosensors is to increase the SNR and DR while minimizing the complexity of both the assay and the detector. Today, this is the main impediment in point-of-care (PoC) biosensors, particularly in high-performance applications such as molecular diagnostics and forensics.
75 citations
TL;DR: This review not only revisits several milestones in the field of nucleic acid-based computation, but also highlights how the prospects for nucleic Acid computation go beyond just a large address space.
57 citations
TL;DR: An orthogonal tryptophanyl tRNA synthetase and tRNA pair is developed, derived from Saccharomyces cerevisiae, that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code.
Abstract: While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.
56 citations
TL;DR: It is shown that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit and provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics.
Abstract: The programmability and replicability of RNA and DNA have respectively enabled the design and selection of a number of allosteric ribozymes and deoxyribozymes. These catalysts have been adapted to function as signal transducers in biosensors and biochemical reaction networks both in vitro and in vivo. However, allosteric control of nucleic acid catalysts is currently limited by the fact that one molecule of effector (input) generally regulates at most one molecule of ribozyme or deoxyribozyme (output). In consequence, allosteric control is usually inefficient when the concentration of input molecules is low. In contrast, catalytic regulation of protein enzymes, as in protein phosphorylation cascades, generally allows one input molecule (e.g., one kinase molecule) to regulate multiple output molecules (e.g., kinase substrates). Achieving such catalytic signal amplification would also be of great utility for nucleic acid circuits. Here we show that allosteric regulation of nucleic acid enzymes can be coupled to signal amplification in an entropy-driven DNA circuit. In this circuit, kinetically trapped DNA logic gates are triggered by a specific sequence, and upon execution generate a peroxidase deoxyribozyme that converts a colorless substrate (ABTS) into a green product (ABTS•+). This scheme provides a new paradigm for the design of enzyme-free biosensors for point-of-care diagnostics.
TL;DR: High‐throughput 454 DNA sequencing of >250,000 VH and Vκ genes from the pre‐ and post‐selection libraries revealed that, in addition to the expected reduction in reading‐frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content.
Abstract: We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.
TL;DR: Phage display is used to identify novel peptides that specifically modulate GlyR function and can readily be adapted for use with other channels that currently lack specific allosteric modulators.
TL;DR: The single-strand siRNA anions were observed to dissociate via cleavage of the 5′ P—O bonds yielding c- and y-type product ions as well as undergo neutral base loss, similar to the dissociation trends observed for duplex DNA.
TL;DR: This unit describes the selection of aptamers that bind to a small molecule target from a single‐stranded RNA pool with two modes of selection, one by column filtration and one by batch selection.
Abstract: This unit describes the selection of aptamers from a single-stranded RNA pool that bind to small molecule targets. Aptamers generated by this type of selection experiment can potentially function as receptors for small molecules in numerous applications, including medical diagnostics, therapeutics, and environmental monitoring. This unit describes two modes of selection, one by column filtration and one by batch selection.
01 Dec 2010
TL;DR: The selection of T7 promoter variants of varying strength are described by emulsifying in vitro transcription with subsequent fluorescence activated cell sorting (FACS) to enrich for active promoters.
Abstract: The ability to build and control complex biological systems is greatly enhanced by the generation of related parts with varying strengths. In this way, various parts can be strung together and the connectivity and expression levels can be matched for the desired system performance. Engineered gene circuits, both in vivo and in vitro, often utilize the T7 RNA polymerase in tandem with the T7 promoter for transcription. In this work, we describe the selection of T7 promoter variants of varying strength by emulsifying in vitro transcription with subsequent fluorescence activated cell sorting (FACS) to enrich for active promoters. Such variant promoters should be of use to synthetic biologists for both in vivo and in vitro applications.
TL;DR: It is shown that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to Ngr, and promote axon elongation of neurons in vitro even in the presence of these inhibitors.
Abstract: Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.
TL;DR: In this paper, the address of X-Chem Incorporated, 100 Beaver Street, Waltham, Massachusetts 02453, USA, was incorrectly given for Keefe. But the correct address is: X-chem Incorporated.
Abstract: Nature Reviews Drug Discovery 9, 537–550 (2010) The address provided for Anthony D. Keefe was incorrect. The correct address is: X-Chem Incorporated, 100 Beaver Street, Waltham, Massachusetts 02453, USA.
TL;DR: This unit describes the selection of aptamers from a pool of single‐stranded RNA by binding to a protein target and can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents.
Abstract: This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. Curr. Protoc. Nucleic Acid Chem. 40:9.3.1-9.3.27. © 2010 by John Wiley & Sons, Inc.
Keywords:
aptamer;
in vitro selection;
affinity reagent;
filter binding assay;
SELEX
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TL;DR: For most of recorded human history, maps had a local focus and were used to navigate from landmark to landmark, but this changed with the invention of the sextant, which quickly became global, although navigation was still largely local.
Abstract: Detailed “fitness landscapes” could reveal the paths for evolution of function. For most of recorded human history, maps had a local focus and were used to navigate from landmark to landmark. This changed with the invention of the sextant, which for the first time allowed us to place ourselves relative to an external standard such as the Sun, Moon, or a distant star. Maps quickly became global, although navigation was still largely local. Then we decided to build our own star-equivalents, satellites, and it became possible to have a Global Positioning System (GPS) that could determine our location to within meters.
22 Dec 2010
TL;DR: A novel epidermal growth factor receptor (EGFR) aptamer biochip is used to identify and isolate cancer cells that overexpress EGFR and these results provide a solid basis for the development of diagnostic devices that can readily identify and isolation CTCs.
Abstract: The isolation and detection of circulating tumor cells (CTCs), when these are few in number or small in mass, can enable early cancer detection [1]. Currently, most research endeavors to isolate circulating tumor cells rely on antibodies and immunohistochemistry [2,3]. Aptamer are alternative molecules that have affinities and specificities that are comparable to those of antibodies [4,5], but these can be more readily adapted to many applications [6–9], including lab-on-a-chip devices. Here, we report use of a novel epidermal growth factor receptor (EGFR) aptamer biochip to identify and isolate cancer cells that overexpress EGFR. These results provide a solid basis for the development of diagnostic devices that can readily identify and isolate CTCs.Copyright © 2010 by ASME