Showing papers by "Andrew D. Ellington published in 2015"
TL;DR: A low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 106 B cells per experiment with demonstrated pairing precision >97%.
Abstract: The determination of immune receptor repertoires using high throughput (NextGen) DNA sequencing has rapidly become an indispensable tool for the understanding of adaptive immunity, antibody discovery and in clinical practice.
324 citations
TL;DR: The asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections.
Abstract: The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.
123 citations
TL;DR: Loop-mediated isothermal amplification of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour, but real-time detection of different genes in multiplex LAMP reactions remains analytically challenging.
Abstract: Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 109 copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex...
122 citations
TL;DR: It is discovered that key mutations in the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia coli gained the ability to grow aerobically on citrate (Cit+ phenotype) during the Lenski long-term evolution experiment.
Abstract: Bacteria and other organisms are constantly under pressure to survive in the face of ever-changing environmental challenges. They generally adapt to these challenges through genetic mutations that modify features they already have. However, occasionally a species may acquire an entirely new characteristic – known as an evolutionary innovation – that allows it to colonize a new environment or adopt a new mode of life. The Lenski Experiment, which began in 1988, is an ongoing study of the evolution of bacteria grown in the laboratory. The experiment started with twelve identical populations of bacteria and has so far tracked the genetic mutations that have been acquired by the populations over tens of thousands of generations. Fifteen years into the experiment, bacteria in one of the populations evolved the ability to exploit a new food source, a molecule called citrate. The bacteria in this population have multiple mutations in a gene called gltA, which encodes an enzyme called citrate synthase. However, it was not clear how these mutations contributed to the ability of the bacteria in this population to use citrate. Here, Quandt et al. have used a variety of genetic and biochemical techniques to examine the mutations in gltA. They found that one mutation occurred before the bacteria evolved the ability to use citrate, and others occurred afterward. The first mutation in gltA increased the activity of the citrate synthase enzyme, which paved the way for a key mutation affecting citrate transport into cells that allowed the bacteria to consume the new food source. However, once the bacteria evolved the ability to use citrate, and more mutations in other genes refined this process, the increased citrate synthase activity became detrimental. At this point, the bacteria acquired a second gltA mutation that lowered citrate synthase activity to a level below what it had been in the original bacteria before the first gltA mutation. The Lenski Experiment presents a rare opportunity to examine the complete history of an evolutionary innovation. Quandt et al. findings show that evolutionary 'reversals' may be necessary to adjust cell processes in different ways as an innovation first evolves and is further refined. A challenge for future work is to identify the other mutations that, together with the first gltA mutation, were necessary for the bacteria to evolve the ability to use citrate.
80 citations
TL;DR: High temperature isothermal amplification method with a thermostable invertase can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto- mole).
Abstract: Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto-mole) Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device
62 citations
TL;DR: This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.
Abstract: The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.
59 citations
TL;DR: The directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter is presented, which can be used to independently control up to six circuits in parallel.
Abstract: T7 RNA polymerase is the foundation of synthetic biological circuitry both in vivo and in vitro due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel.
57 citations
TL;DR: It is shown that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range, and the resulting polymerase mutants can be used to generate 2′-O-methyl modified RNA with yields much higher than enzymes currently employed.
Abstract: On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.
57 citations
TL;DR: An E. coli tRNA(Sec) that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency is evolved and allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.
Abstract: Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNASec needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNASec that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNASec allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.
56 citations
TL;DR: It is shown that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138+ antibody-secreting cells within the PLN, and an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals is developed.
Abstract: The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138(+) antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.
46 citations
TL;DR: A paper-based assay platform for detection of ricin a chain based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich is reported, which was detected at concentrations as low as 34 pM.
Abstract: We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min.
TL;DR: Developing RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy and site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable.
Abstract: HIV-1 aspartyl protease (PR) plays a key role in virion morphogenesis, underscoring the effectiveness of protease inhibitors (PI). Despite their utility, side effects and drug-resistance remains a problem. We report the development of RNA aptamers as inhibitors of HIV-1 PR for potential use in anti-HIV gene therapy. Employing Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated four unique families of anti-HIV-1 PR RNA aptamers displaying moderate binding affinities (Kd = 92–140 nmol/l) and anti-PR inhibitory activity (Kis = 138–647 nmol/l). Second-generation RNA aptamers selected from partially randomized pools based on two of the aptamer sequences displayed striking enhancements in binding (Kds = 2–22 nmol/l) and inhibition (Kis = 31–49 nmol/l). The aptamers were specific in that they did not bind either the related HIV-2 protease, or the cellular aspartyl protease, Cathepsin D. Site-directed mutagenesis of a second-generation aptamer to probe the predicted secondary structure indicated that the stem-loops SL2 and SL3 and the stem P1 were essential for binding and that only the 3'-most 17 nucleotides were dispensable. Anti-PR aptamers inhibited HIV replication in vitro and the degree of inhibition was higher for second-generation aptamers with greater affinity and the inhibition was abrogated for a nonbinding aptamer variant.
TL;DR: The results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acids circuitry.
Abstract: An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.
TL;DR: The results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline, which leads to diversified phosphorylation states ofCTD in vitro and in cells.
Abstract: Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.
TL;DR: A focus on expanded chassis and end-to-end design in an effort to move beyond the use of E. coli and S. cerivisiea to organisms better suited to fermentation and production is highlighted.
Abstract: The advancement of synthetic biology over the past decade has contributed substantially to the growing bioeconomy. A recent report by the National Academies highlighted several areas of advancement that will be needed for further expansion of industrial biotechnology, including new focuses on design, feedstocks, processing, organism development, and tools for testing and measurement; more particularly, a focus on expanded chassis and end-to-end design in an effort to move beyond the use of E. coli and S. cerivisiea to organisms better suited to fermentation and production; second, continued efforts in systems biology and high-throughput screening with a focus on more rapid techniques that will provide the needed information for moving to larger scale; and finally, work to accelerate the building of a holacratic community with collaboration and engagement between the relevant government agencies, industry, academia, and the public.
TL;DR: A combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species, and presents an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures.
Abstract: Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat that they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. For example, a human single nucleotide polymorphism (SNP) in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable cocrystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step toward computationally predicting the “host jumping” potential of a virus into a new host species. IMPORTANCE We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing the potential spillover risks of viruses circulating in animal populations.
TL;DR: The authors' models indicate that these genetic circuits that allow trading of a common good required for growth in bacteria can demonstrate comparative advantage, and that cooperation in such a manner is particularly favored under stringent external conditions and when the cost of production is not overly high.
Patent•
17 Sep 2015TL;DR: In this paper, compositions and methods for isothermal nucleic acid amplification and detection are described and discussed. But none of the methods are suitable for the detection of nucleic acids.
Abstract: Disclosed are compositions and methods for isothermal nucleic acid amplification and detection.
TL;DR: RecA, a recombination enzyme that catalyzes sequence specific strand exchange, can be used to increase circuit rates up to 9-fold, improving signal-to-noise and the introduction of RNA into DNA circuits further controls the specificity of RecA strand exchange.
23 Dec 2015
TL;DR: A one-step tumor cell detection approach based on the dynamic morphological behavior tracking of cancer cells on a ligand modified surface that provided feature vectors that were statistically distinct between normal and cancer cells.
Abstract: In this paper, we report a one-step tumor cell detection approach based on the dynamic morphological behavior tracking of cancer cells on a ligand modified surface. Every cell on the surface was tracked in real time for several minutes immediately after seeding until these were finally attached. Cancer cells were found to be very active in the aptamer microenvironment, changing their shapes rapidly from spherical to semi-elliptical, with much flatter spread and extending pseudopods at regular intervals. When incubated on a functionalized surface, the balancing forces between cell surface molecules and the surface-bound aptamers, together with the flexibility of the membranes, caused cells to show these distinct dynamic activities and variations in their morphologies. On the other hand, healthy cells remained distinguishingly inactive on the surface over the same period. The quantitative image analysis of cell morphologies provided feature vectors that were statistically distinct between normal and cancer cells.
TL;DR: The use of Spinach.ST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications and the same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans.
Abstract: We describe design parameters for the synthesis and analytical application of a label-free RNA molecular beacon, termed SpinachST The RNA aptamer Spinach fluoresces upon binding the small-molecule fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) Spinach has been reengineered by extending its 5′- and 3′-ends to create SpinachST, which is predicted to fold into an inactive conformation that fails to bind DHFBI Hybridization of a trigger oligonucleotide to a designed toehold on SpinachST initiates toehold-mediated strand displacement and restores the DFHBI-binding, fluorescence-enhancing conformation of Spinach The versatile SpinachST sensor can detect DNA or RNA trigger sequences and can readily distinguish single-nucleotide mismatches in the trigger toehold Primer design techniques are described that augment amplicons produced by enzymatic amplification with SpinachST triggers Interaction between these triggers and SpinachST molecular beacons leads to the real-time, sequence-specific quantitation of these amplicons The use of SpinachST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications The same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans
TL;DR: A new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array, and should be applicable to any mixture of complex and subtly different analytes for whichucleic acid-based receptors exist.
Abstract: Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi-specific receptors, Next-Gen DNA sequencing was used to generate data, and cell line differentiation was the test-bed application. The study of a principal component analysis loading plot revealed cross-reactivity between the aptamers. The technique generates high-dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid-based receptors exist.
Patent•
15 Sep 2015TL;DR: In this article, a method for identifying proteins and peptides, and more specifically large-scale sequencing of single peptides in a mixture of diverse peptides at the single molecule level, was proposed.
Abstract: The present invention relates to the field of identifying proteins and peptides, and more specifically large-scale sequencing of single peptides in a mixture of diverse peptides at the single molecule level. The present invention also relates to methods for identifying amino acids in peptides, including peptides comprising unnatural amino acids. In one embodiment, the present invention contemplates labeling the N-terminal amino acid with a first label and labeling an internal amino acid with a second label. In some embodiments, the labels are fluorescent labels. In other embodiments, the internal amino acid is Lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.
Patent•
13 Feb 2015TL;DR: In this paper, a hairpin and a single strand extension at the 3' end are employed to identify a single nucleotide polymorphism in a particular sequence, where at least one nucleotide is mismatched compared to a target particular sequence.
Abstract: Provided herein are compositions and methods for identification of the presence or absence of a particular sequence, such as a single nucleotide polymorphism. Employed herein are particular primers that comprise a hairpin and a single strand extension at the 3' end, the single strand extension in which at least one nucleotide is mismatched compared to a target particular sequence. Strand displacement that leads to additional binding of the primer and extension of the primer occurs following initial binding of the primer to the nucleic acid comprising the particular sequence.
15 Sep 2015
TL;DR: In this article, a method for identifying amino acids in peptides, including peptides with one or more unnatural amino acids, was proposed, based on the fluorescent signature for each peptide at the single molecule level.
Abstract: Identifying proteins and peptides, and more specifically large-scale sequencing of single peptides in a mixture of diverse peptides at the single molecule level is an unmet challenge in the field of protein sequencing. Herein are methods for identifying amino acids in peptides, including peptides with one or more unnatural amino acids. In one embodiment, the N-terminal amino acid is labeled with a first label and an internal amino acid is labeled with a second label. In some embodiments, the labels are fluorescent labels. In other embodiments, the internal amino acid is Lysine. In other embodiments, amino acids in peptides are identified based on the fluorescent signature for each peptide at the single molecule level.
TL;DR: A pair of artificial DNA bases have been shown to adopt an edge-to-edge geometry in DNA which is similar that found in Watson–Crick base pairing, which allows aptamers containing these bases to bind more strongly to a target than those developed using only the four naturally occurring bases.
Abstract: A pair of artificial DNA bases have now been shown to adopt an edge-to-edge geometry in DNA which is similar that found in Watson–Crick base pairing. Aptamers containing these bases have also been shown to bind more strongly to a target than those developed using only the four naturally occurring bases.
TL;DR: It is demonstrated that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation.
TL;DR: The development of directed evolution methods for nucleic acids was an idea whose time had come, as evidenced by its multiple, independent origins, and the discovery of the in vitro selection of aptamers/SELEX is often cited as a milestone in directed evolution.
Abstract: The development of directed evolution methods for nucleic acids was an idea whose time had come, as evidenced by its multiple, independent origins. It can be argued that Oliphant and Struhl were some of the earliest practitioners, having randomized oligonucleotides to identify binding promoters and repressor binding sites in cells, circa 1986–1987 (Oliphant et al. 1989, 1986; Oliphant and Struhl 1987, Oliphant and Struhl 1988). Just as the GCN4 repressor was used to capture binding sites by affinity chromatography (Oliphant et al. 1989), Blackwell and Weintraub (1990) used electrophoretic mobility shift assays to select for differential binding by homoand heterooligomers of the helix-loop-helix proteins MyoD and E2A. Robertson and Joyce (1990) were amongst the first to recognize that randomization and selection could be applied to functional nucleic acids, such as the Tetrahymena self-splicing ribozyme, although I have always argued that this honor might first go to Sol Spiegelman, whose ideas were far in advance of the technologies of his time. As early as 1970, the Spiegelman lab was able to remarkably select for Qb ‘mini monster’ variants whose in vitro replication was resistant to ethidium bromide (Saffhill et al. 1970). Just as the Szostak lab of the early 1990s was a remarkable place to be (Doudna, Green, Bartel, Famulok), the Spiegelman lab of the late 1960s and early 1970s must have been extraordinary (Mills, Pace, Kramer, with Orgel as a collaborator). That said, the discovery of the in vitro selection of aptamers/SELEX is often cited as a milestone in directed evolution. Tuerk and Gold (1990) selected a non-natural protein-binding sequence. The Szostak lab selected a nonnatural dye-binding sequence (Ellington and Szostak 1990). The Gold lab used a library of 8 random nucleotides; the Szostak lab used a library of 100 random nucleotides. The Gold lab patented their findings; the Szostak lab did not. The rest, as they say, is history. A potentially interesting question is: Why was the selection of dye-binding aptamers exciting, given the previous discovery of ethidium bromide not-binding Qb replicators? A portion of the answer of course has to do with the extraordinary 20 years displacement of the first and second findings. We stand on the shoulders of giants, and then quickly forget their names. The advances in technology in that 20 years period are another rationale: in vitro selection/SELEX became possible because of advances in synthetic DNA technology (making library production possible) and molecular amplification technologies (PCR). The expanse in capability is like the wondrous transformation of highly automated and parallel sequencing into NextGen sequencing. Both are still sequencing, but the scale is now staggering. But I think the most significant difference between the two findings was the hint that there was an undiscovered country in deep sequence space (to further mix Shakespearian and Star Trek metaphors). The Big Idea that biology is but a subset of a larger set of possibilities is still mind-blowing. Crick (1968) talked about the frozen accident of the genetic code, but the foment at origins that led to something capable of being frozen is still largely unimaginable. That Big Idea got a little bit lost along the way, with directed evolution being used for many biotechnology applications, but often only in passing to & Andrew D. Ellington ellingtonlab@gmail.com
20 Mar 2015
TL;DR: In this article, mutations that increase the transcriptional activity of the variants with broad substrate range are presented, with a focus on T7 RNA polymerase variants with enhanced transcriptional activities.
Abstract: Disclosed are T7 RNA polymerase variants with enhanced transcriptional activity. T7 RNA polymerase variants are known which have the ability to incorporate modified ribonucleotides into growing RNA molecules. However, these variants have relatively low levels of transcriptional activity. Presented herein are mutations that increase the transcriptional activity of the variants with broad substrate range.