Random Library Research Articles

High efficiency transformation and mutant screening of the laurel wilt pathogen, Raffaelea lauricola.

The fungal pathogen, Raffaelea lauricola, is the causative agent of laurel wilt, a devastating disease affecting the Lauraceae family. The fungus is vectored by ambrosia beetles that carry the fungus in specialized structures (mycangia), with the fungus acting as a symbiont and food source for beetle larvae growing in tree galleries. In order to probe the molecular basis for plant pathogenicity and insectsymbiosis of the laurel wilt fungus, molecular tools including establishment of efficient transformation protocols are required. Resistance marker profiling revealed susceptibility of R. lauricola to phosphinothricin, chlorimuron ethyl, hygromycin, and benomyl. Agrobacterium-mediated transformation using either the bar or sur marker resulted in 1-200 transformants/105 spores. A second protocol using lithium acetate-polyethylene glycol (LiAc-PEG) treatment of fungal blastospores yielded 5-60 transformants/μg DNA/108 cells. Transformants were mitotically stable (at least 5 generations), and > 95% of transformants showed a single integration event. R. lauricola strains expressing green and red fluorescent proteins (EGFP and RFP), as well as glucuronidase (GUS), were constructed. Using the Agrobacterium-mediated method, a random T-DNA insertion library was constructed, and genetic screens led to the isolation of developmental mutants as well as mutants displaying enhanced resistance to sodium dodecyl sulfate (SDS) or fluconazole, and those showing decreased susceptibility to biphenol. These results establish simple and reliable genetic tools for transformation of R. lauricola needed for genetic dissection of the symbiotic and virulent lifestyles exhibited by this fungus and establish a library of insertion mutants that can be used in various genetic screens to dissect molecular pathways. KEY POINTS: • Vectors and transformation protocols were developed for Raffaelea lauricola. • Method was used for construction of a random insertion mutant library. • Mutant library was validated by phenotypic screens for resistance and susceptibility to various agents.

Landscape of in vivo Fitness-Associated Genes of Enterobacter cloacae Complex.

Species of the Enterobacter cloacae complex (ECC) represent an increasing cause of hospital-acquired infections and commonly exhibit multiple antibiotic resistances. In order to identify genes that may play a role in its ability to colonize the host, we used the transposon-sequencing (Tn-seq) approach. To this end, a high-density random transposon insertion library was obtained from E. cloacae subsp. cloacae ATCC 13047, which was used to analyze the fitness of ca. 300,000 mutants in Galleria mellonella colonization model. Following massively parallel sequencing, we identified 624 genes that seemed essential for the optimal growth and/or the fitness within the host. Moreover, 63 genes where mutations resulted in positive selection were found, while 576 genes potentially involved in the in vivo fitness were observed. These findings pointed out the role of some transcriptional regulators, type VI secretion system, and surface-associated proteins in the in vivo fitness of E. cloacae ATCC 13047. We then selected eight genes based on their high positive or negative fold changes (FCs) and tested the corresponding deletion mutants for their virulence and ability to cope with stresses. Thereby, we showed that ECL_02247 (encoding the NAD-dependent epimerase/dehydratase) and ECL_04444 (coding for a surface antigen-like protein) may correspond to new virulence factors, and that the regulator ECL_00056 was involved in in vivo fitness. In addition, bacterial cells lacking the flagellum-specific ATP synthase FliI (ECL_03223) and the hypothetical protein ECL_01421 were affected for mobility and resistance to H2O2, respectively. All these results yield valuable information regarding genes important for infection process and stress response of E. cloacae ATCC 13047 and participate to a better understanding of the opportunistic traits in this bacterial pathogen.

Potential Use of Antifreeze DNA Aptamers for the Cryopreservation of Human Erythrocytes

This article summarizes proof of concept experiments which clearly demonstrated the ability of DNA aptamers selected against a molecular mimic of early ice crystal nuclei to protect the integrity of human erythrocytes following a slow freeze-thaw cycle. Following 10 cycles of selection and DNA amplification of rare candidate DNA aptamers against a copper-organic ligand complex which holds water molecules in a conformation resembling early ice crystal nuclei, the aptamers were tested for their ability to preserve erythrocyte morphology by phase-contrast microscopy versus controls without aptamers and with the original randomized aptamer DNA library template following slow freezing and storage overnight at -20 °C with slow thawing at 25 °C. Those experiments revealed that a minimum of 32 μg/ml of the final selected aptamer pool of DNA molecules was required to completely protect nearly 100% of the erythrocytes. By contrast, the treatment groups without the aptamers or with the randomized aptamer template DNA at 32 μg/ml produced only fragmented erythrocytes and cellular debris (no intact cells), thus indicating that specifically selected DNA conformations were required to bind and limit the size of forming ice crystals to cryoprotect the erythrocytes.

Aptamer-linked immobilized sorbent assay for detecting GMO marker, phosphinothricin acetyltransferase (PAT)

Development of genetically modified crops has rapidly increased in last few years. The most widely grown GM crops express genes that confer herbicide tolerance and insect resistance. Detection system of GM crops is important for safety evaluation before its consumption. The purpose of this research is to detect GM crops, especially PAT, in food-samples. The bar gene (PAT protein, herbicide resistant) was cloned in pGEX-4T-1 and expressed by E. coli. The high-affinity PAT-specific single-stranded DNA (ssDNA) aptamers were obtained from a random DNA library. MOE docking study was performed to identify the potential binding region of the selected aptamers on PAT. Aptamer-linked immobilized sorbent assay (ALISA) method was used to detect PAT. We screened aptamer against PAT for developing an efficient detection method. The selected PAT specific aptamers, HRPA-05 and HRPA-07, showed the distinct target binding behaviors, and detected PAT protein by aptamer-linked immobilized sorbent assay method with high efficiency and selectivity.

Extrapolation of mortality in COVID-19: Exploring the role of age, sex, co-morbidities and health-care related occupation.

We used a publicly available data of 44,672 patients reported by China's centre for disease control to study the role of age, sex, co-morbidities and health-care related occupation on COVID-19 mortality. The data is in the form of absolute numbers and proportions. Using the percentages, retrospective synthetic data of 100 survivors and 100 deaths were generated using random number libraries so that proportions of ages, genders, co-morbidities, and occupations were constant as in the original data. Logistic regression of the four predictor factors of age, sex, co-morbidities and occupation revealed that only age and comorbidities significantly affected mortality. Sex and occupation when adjusted for other factors in the equation were not significant predictors of mortality. Age and presence of co-morbidities correlated negatively with survival with co-efficient of -1.23 and -2.33 respectively. Odds ratio (OR) for dying from COVID-19 for every 10-year increase in age was 3.4 compared to the previous band of 10 years. OR for dying of COVID-19 was 10.3 for the presence of any of the co-morbidities. Our findings could help in triaging the patients in the emergency room and emphasize the need to protect the elderly and those with comorbidities from getting exposed.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches – formerly indicated to be involved in CPR:CYP interactions – or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Optimized methodology for product recovery following emulsion PCR: applications for amplification of aptamer libraries and other complex templates.

Bias and background issues make efficient amplification of complex template mixes such as aptamer and genomic DNA libraries via conventional PCR methods difficult; emulsion PCR is being increasingly used in such scenarios to circumvent these problems. However, before products generated via emulsion PCR can be used in downstream workflows, they need to be recovered from the water-in-oil emulsion. Often, emulsions are broken following amplification using volatile organic solvents, and product is subsequently isolated via precipitation. Unfortunately, the use of such solvents requires the implementation of special environmental controls, and the yield and purity of DNA isolated by precipitation can be highly variable. Here, we describe the optimization of a simple protocol which can be used to recover products following emulsion PCR using a 2-butanol extraction and subsequent DNA isolation via a commercially available clean-up kit. This protocol avoids the use of volatile solvents and precipitation steps, and we demonstrate that it can be used to reliably recover DNA from water-in-oil emulsions with efficiencies as high as 90%. Furthermore, we illustrate the practical applicability of this protocol by demonstrating how it can be implemented to recover a complex random aptamer library following amplification via emulsion PCR.

Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system

Pleurotus eryngii wild-type versatile peroxidase (wtVP) oxidizes structurally diverse substrates in an H2O2-dependent manner, but its ability to oxidize many pollutants is limited by suicidal enzyme inactivation in the presence of excess H2O2. To address this drawback, we generated random mutagenesis libraries containing 3 × 106 mutated VP genes and screened for enzymes with higher oxidative stability expressed on the surface of yeast cells. This was achieved by flow cytometry using the substrate fluorescein tyramide. After two rounds of sorting, the percentage of cells expressing variants with improved oxidative stability had increased from 1 % to 56 %. The most stable variants featured 3–5 amino acid substitutions and retained up to 70 % of their initial activity after incubation for 1 h in 30 mM H2O2 (conditions that completely inactivate wtVP). Selected variants were extracted from yeast cell walls and purified for kinetic characterization. We also prepared yeast cell walls with wtVP and the three most stable VP variants for multiple cycles of azo dye (Reactive black 5) degradation. After 10 cycles of 12 h, two of the variants retained more than 97 % of their initial activity, whereas the activity of wtVP declined by ∼30 %. These results confirm that our high-throughput screening system can improve the oxidative stability of versatile peroxidase, providing a source of novel enzymes for remediation applications.

Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae.

In this study, the prokaryotic expression system of Escherichia coli was used to modify prolyl aminopeptidase derived from Aspergillus oryzae JN-412 (AoPAP) via random mutagenesis and site-directed saturation mutagenesis. A random mutant library with a capacity of approximately 3000 mutants was compiled using error-prone polymerase chain reaction, and nonconservative amino acids within 3Å of the substrate L-proline-p-nitroaniline were selected as site-directed saturation mutagenesis sites via homologous simulation and molecular docking of AoPAP. Variants featuring high catalytic efficiency were screened by a high-throughput screening method. The specific activities of the variants of 3D9, C185V, and Y393W were 127Umg-1, 156Umg-1, and 120Umg-1, respectively, which were 27%, 56%, and 20% higher than those of the wild type, with a value of 100Umg-1. The half-life of thermostability of the mutant 3D9 was 4.5h longer than that of the wild type at 50°C. The mutant C185V improved thermostability and had a half-life 2h longer than that of the wild type at a pH of 6.5. Prolyl aminopeptidase had improved stability within the acidic range and thermostability after modification, making it more suitable for a synergistic combination with various acidic and neutral endoproteases.

Discovery of sulfonamides and 9-oxo-2,8-diazaspiro[5,5]undecane-2-carboxamides as human kynurenine aminotransferase 2 (KAT2) inhibitors

Human kynurenine aminotransferase 2 (KAT2) inhibitors could be potentially used to treat the cognitive deficits associated with bipolar disease and schizophrenia. Although, there has been active drug research activity by several industrial and academic groups in developing KAT2 inhibitors over the years, no such compound has proceeded to the clinics. Here, we report two different chemical series of reversible KAT2 inhibitors with sub-micromolar activities. The first series was identified by a high-throughput screening of a diverse random library and the second one by structure-based virtual screening. Two novel crystal structures of KAT2 complexed with different reversible inhibitors were also deposited to the Protein databank which could be useful for future drug discovery efforts.

Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability

Lignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30mMH2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1h incubation in 30mMH2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.

Targeting epimastigotes of Trypanosoma cruzi with a peptide isolated from a phage display random library

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.

Speculations on Eudora Welty's Reading of Seven Gothic Tales and Out of Africa by Isak Dinesen

Speculations on Eudora Welty's Reading of Seven Gothic Tales and Out of Africa by Isak Dinesen Pearl Amelia McHaney Eudora Welty reviewed Isak Dinesen's 1957 Last Tales and two scholarly studies of the Danish writer's oeuvre for the New York Times Book Review. She read, and passed on to William Maxwell, Dinesen's essay "On Mottoes of My Life," and she also contributed to a memorial tribute for Dinesen (the pen name of Karen Blixen) in 1965. These commentaries are part of Welty's oeuvre and have been reprinted and made available to readers and scholars. Until 2019, the first known book review that Welty had published was of Marguerite Steedman's But You'll Be Back for the Saturday Review of Literature in 1942. Recently, however, I learned that in 1938 Welty had reviewed Dinesen's memoir Out of Africa in her hometown paper, the Jackson Clarion Ledger.1 In her review, Welty also acknowledges having read Seven Gothic Tales, Dinesen's first book. By 1938, when she first reviewed Dinesen's work, Welty had traveled throughout Mississippi for her five months as a junior publicity agent for the Works Progress Administration and had had one photography exhibit in the Jackson Municipal Art Gallery and two photography exhibits in New York City. She had published eleven short stories in truly "little" magazines and had written and burned the first version of "Petrified Man." Edward O'Brien had reprinted "Lily Daw and the Three Ladies" in his edition of The Best Short Stories of 1938. Welty was eighteen months away from receiving Diarmuid Russell's invitation to be her primary reader and agent. She was years away from writing a novel, and nearly a half-century would pass before she would write her own memoir. To write a book review is to read closely and then to think, almost from afar, of which details and which impressions to gather and share. To review Out of Africa, the second book by Dinesen, Welty begins by commenting that "'Seven Gothic Tales' appeared several years ago under the name Isak Dinesen. It was a widely read book of ornate, metaphysical stories designed carefully and with a sharp obviously feminine humor, to delight and tantalize the reader who likes curious and unreal things" (1). This brief summary of Dinesen's first book illustrates, most importantly, that Welty had read Seven Gothic Tales prior to writing her Out of Africa review, and, in addressing the "reader who likes curious and unreal things," she begins [End Page 125] the habit of reviewing from a reader's perspective—a point of view she maintains in nearly all of her book reviews. The library of books in Welty's home includes the 1934 Random House Modern Library edition of Seven Gothic Tales that reprints the Smith and Haas publication with Dorothy Canfield's introduction. This first collection of Dinesen's tales was an April Book-of-the-Month Club selection and was on the New York Times Best Seller list for April, May, June, and July of 1934. The book is a complex exploration of the Gothic: sudden dark, stormy, windy weather causing drastic consequences; ancient worlds bearing down on the present through elaborate allusions; corporeal ghosts and shadowy people; unexpected, fantastic events; death; strange visages and bodies; and riddles posed to the characters and readers. Dinesen asks, "'What is it,' she said very slowly, in the manner of a sibylla, 'which is bought dearly, offered for nothing, and then most often refused?'" (Seven 115).2 Peter Monro Jack reviewed Seven Gothic Tales in the New York Times Book Review under the headline "A Strange Book for Our Time." Jack found the tales "curious" with an "almost Proustian sensibility that illuminates, by refining, the romantic mood." Citing that Dinesen's "pattern is as flexible as Scheherazade's," Jack asserts that Dinesen's "inventiveness is apparently inexhaustible," and he concludes, "This is a work of the imagination, very subtle and graceful; a rare and curious book, it will be white magic to many and mere mummery to others … though both are in the book, there is more magic than mummery" (6). This evaluation, I believe, would...

Application and development of aptamer in cancer: from clinical diagnosis to cancer therapy.

Traditional anticancer therapies can cause serious side effects in clinical treatment due to their nonspecific of tumor cells. Aptamers, also termed as 'chemical antibodies', are short DNA or RNA oligonucleotides selected from the synthetic large random single-strand oligonucleotide library by systematic evolution of ligands by exponential enrichment (SELEX) to bind to lots of different targets, such as proteins or nucleic acid structures. Aptamers have good affinities and high specificity with target molecules, thus may be able to act as drugs themselves to directly inhibit the proliferation of tumor cells, or own great potentialities in the targeted drug delivery systems which can be used in tumor diagnosis and target specific tumor cells, thereby minimizing the toxicity to normal cells. Here we review the unique properties of aptamer represents a great opportunity when applied to the rapidly developing fields of biotechnology and discuss the recent developments in the use of aptamers as powerful tools for analytic, diagnostic and therapeutic applications for cancer.

Evaluation of different direct and indirect SELEX monitoring methods and implementation of melt-curve analysis for rapid discrimination of variant aptamer sequences.

Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an iterative method for in vitro selection of aptamers from a random synthetic oligonucleotide library. Successful retrieving of aptamers by SELEX relies on optimization of various steps including target immobilization, aptamer partitioning, amplification, and ssDNA generation, which all require spending considerable effort and cost. Furthermore, due to the random nature of the initial library, SELEX may redirect toward the selection of low-affinity aptamers that are over-represented in the ssDNA population due to PCR bias. Thus, precise monitoring of the SELEX process is crucial to ensure the selection of target-specific aptamers. In the present study, we investigated the reliability and simplicity of different direct and indirect monitoring methods including UV-Vis spectroscopy, real-time PCR quantification and melt-curve analysis, electrophoretic mobility shift assay (EMSA) and enzyme-linked oligonucleotide assay (ELONA) for selection of DNA aptamers for a protein target. All the examined methods were capable of illustrating the gradual evolution of specific aptamers by the progression of SELEX and showed almost similar results regarding the identification of the enriched round of selection. Moreover, we describe the use of melt-curve analysis in the colony real-time PCR method as a simple, robust, and repeatable tool for pre-sequencing separation of distinct aptamer clones.

Creating a Library of Random Transposon Mutants in Leptospira.

Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli β2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.