Random Oligonucleotide Research Articles

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.

Nucleus Accumbens-Associated Protein 1 Binds DNA Directly through the BEN Domain in a Sequence-Specific Manner.

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short β sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

Organocatalytic strategy to telechelic oligo(ε-caprolactone-co-p-dioxanone): Photocurable macromonomers for polyester networks

We have designed photocurable, telechelic macromonomers consisting of random oligo(ε-caprolactone-co-p-dioxanone), oligo(CL-co-DX), and demonstrated their suitability for preparing pliable polyester networks whose properties resemble those of gels.A versatile and effective metal-free co-oligomerization, catalyzed by diphenyl phosphate, was developed in bulk and at room temperature. A high rate of conversion of monomers was achieved and oligo(CL-co-DX)s with different composition and topology were obtained with controlled molar mass, approx. 2000 g mol−1, low dispersity and a random distribution of the two monomeric units. Kinetics analysis of the reaction disclosed a faster incorporation rate for the p-dioxanone (DX) than ε-caprolactone (CL). The extrapolated rate constant, kDX, was 0.030 min−1 against a kCL of 0.013 min−1. The reactivity ratios were respectively 2.7 (rDX) and 0.28 (rCL). A detailed NMR analysis was performed to elucidate the structure of the co-oligomers, which could be precisely controlled by varying the monomer feed ratio and initiator. Depending on the composition, amorphous to semicrystalline oligomers with melting points close to room temperature were obtained, which after acrylation of the chain-end gave polyester networks with high swelling capacity up to 700%, and water uptake up to 70%.

Genetic Diversity of Brinjal Fruit and Shoot Borer (BSFB) Population of Odisha, India

In Southern and Southern East Asia, the eggplant fruit and shoot borer (BSFB) is the most harmful pest for eggplant. The pest is known to cause about 16 and 70% damage to shoots and fruits, respectively. It is of practical importance to precisely trace the genetic variability in the expanding geographical distribution of the pest. In the current study, the genetic variability was analysed among geographically confined populations of BSFB from thirty locations of Odisha adopting RAPD-PCR technique. Twenty random 10-mer oligonucleotide primers were cast off to amplify genomes of thirty BSFB populations. A total of 118 bands were spotted by 10 primers, of which 116 were polymorphic. Thirteen unique bands were recognized which will be helpful for developing diagnostic markers. Genetic similarity among BSFB populations ranges from 0.37 to 0.93, with a mean of 0.65 specifying that wide genetic variation showed among BSFB populations at molecular level. Most of the populations could be, especially prominent from each other and clustered into two major clusters. The Structure software indicated a significant genetic differentiation among the populations. More research with host differentials can establish their biotype status.

The Role of RNA and DNA Aptamers in Glioblastoma Diagnosis and Therapy: A Systematic Review of the Literature

Glioblastoma (GBM) is the most lethal primary brain tumor of the central nervous system in adults. Despite advances in surgical and medical neuro-oncology, the median survival is about 15 months. For this reason, initial diagnosis, prognosis, and targeted therapy of GBM represent very attractive areas of study. Aptamers are short three-dimensional structures of single-stranded nucleic acids (RNA or DNA), identified by an in vitro process, named systematic evolution of ligands by exponential enrichment (SELEX), starting from a partially random oligonucleotide library. They bind to a molecular target with high affinity and specificity and can be easily modified to optimize binding affinity and selectivity. Thanks to their properties (low immunogenicity and toxicity, long stability, and low production variability), a large number of aptamers have been selected against GBM biomarkers and provide specific imaging agents and therapeutics to improve the diagnosis and treatment of GBM. However, the use of aptamers in GBM diagnosis and treatment still represents an underdeveloped topic, mainly due to limited literature in the research world. On these bases, we performed a systematic review aimed at summarizing current knowledge on the new promising DNA and RNA aptamer-based molecules for GBM diagnosis and treatment. Thirty-eight studies from 2000 were included and investigated. Seventeen involved the use of aptamers for GBM diagnosis and 21 for GBM therapy. Our findings showed that a number of DNA and RNA aptamers are promising diagnostic and therapeutic tools for GBM management.

Accumulated data and results from the recent study of dsRNA isolated from grapevines used in experiments of insect and graft transmission of 'Shiraz' disease

RT-PCR amplicons of dsRNA isolated from various grapevines, which were used in the experiments of transmission of 'Shiraz' disease (SD) from 'Cinsaut Blanc' clone P163/12 to SD-susceptible 'Merlot' and 'Shiraz' using mealybug Planococcus ficus and grafting were investigated. The amplicons were generated in RT-PCR based on virus-specific or random hexamers oligonucleotide primers. Standard molecular techniques and high-throughput sequencing (HTS), respectively, were applied. The results supported the hypothesis that GVA M5v variant present in 'Cinsaut Blanc' P163/12, which is a member of group II of GVA variants associated with SD, is crucial for developing this disease. HTS data did not reveal any other grapevine viruses besides GLRaV-3 and GVA in SD-affected grapevines, except for GVE which, however, was not present in all diseased plants.

Analysis of Modified Nucleotide Aptamer Library Generated by Thermophilic DNA Polymerases.

One of the pivotal steps in aptamer selection is the amplification of target‐specific oligonucleotides by thermophilic DNA polymerases; it can be a challenging task if nucleic acids possessing modified nucleotides are to be amplified. Hence, the identification of compatible DNA polymerase and modified nucleotide pairs is necessary for effective selection of aptamers with unnatural nucleotides. We present an in‐depth study of using 5‐indolyl‐AA‐dUTP (TAdUTP) to generate oligonucleotide libraries for aptamer selection. We found that, among the eight studied DNA polymerases, only Vent(exo‐) and KOD XL are capable of adapting TAdUTP, and that replacing dTTP did not have a significant effect on the productivity of KOD XL. We demonstrated that water‐in‐oil emulsion PCR is suitable for the generation of aptamer libraries of modified nucleotides. Finally, high‐throughput sequence analysis showed that neither the error rate nor the PCR bias was significantly affected by using TAdUTP. In summary, we propose that KOD XL and TAdUTP could be effectively used for aptamer selection without distorting the sequence space of random oligonucleotide libraries.

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

Lesional Inflammatory Profile in Hidradenitis Suppurativa Is Not Solely Driven by IL-1

A synthetic Pur-based peptide binds and alters G-quadruplex secondary structure present in the expanded RNA repeat of C9orf72 ALS/FTD

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60–80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD.

A Model for the Emergence of RNA from a Prebiotically Plausible Mixture of Ribonucleotides, Arabinonucleotides, and 2'-Deoxynucleotides.

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2′-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2′-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2′-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2′-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Oligonucleotide-induced regulation of the oxidase-mimicking activity of octahedral Mn3O4 nanoparticles for colorimetric detection of heavy metals.

A colorimetric assay for the determination of heavy metal ions is presented that is based on the regulation of the oxidase-mimicking activity of Mn3O4 nanoparticles (NPs) by oligonucleotides. The chromogenic agent tetramethylbenzidine (TMB) is oxidized by the catalytic action of Mn3O4 NPs to generate products that have a yellow color in acidic solution, with a peak at 450nm. It is found that random oligonucleotides are absorbed on the regular surface of the Mn3O4 NPs and temporarily inhibit the oxidation of TMB. This leads to a decrease in absorbance and a light-green coloration of the solution. The results show that the purine bases in oligonucleotides play a key role in their regulation of the activity of the NPs. The regulatory effect is assumed to be of the noncompetitive type. In the presence of heavy metal ions like Hg(II) or Cd(II), the inhibition is canceled due to the binding of heavy metal ions to thymine bases, and the color of the solution changes from light green to yellow. The increase in absorbance at 450nm is related to the amount of heavy metal ions present. The method allows Hg(II) and Cd(II) to be determined visually in concentrations as low as 20μg·L-1. The detection limit of the colorimetric assay is 3.8 and 2.4μg·L-1 of Hg(II) and Cd(II), respectively. The assay displays good selectivity over other heavy metal ions. The method was successfully validated by analyzing several water samples. Graphical abstract Schematic representation of the colorimetric assay for Hg(II) and Cd(II) based on the intrinsic oxidase-mimicking activity of Mn3O4 nanoparticles that isregulated by oligonucleotides.

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.

RAPD-PCR analysis and gene expression of CYP450 in Tribolium castaneum adults in response to different insecticides

Random amplified polymorphic DNA analysis by polymerase chain reaction (RAPD-PCR) was used as a tool to screen genetic variation in Tribolium castaneum adults treated by LC50 of Bacillus thuringienisis, Beauveria bassiana, teflubenzuron and spinetoram using ten random oligonucleotide primers for amplification. The results of cluster analysis using Dice coefficient among treated and untreated T. castaneum adults revealed that adult beetles were grouped into 2 main clusters. One contains control adults. The other cluster was divided into 2 main subclusters; one includes adults treated with LC50 of B. thuringienisis and teflubenzuron while the other subcluster contains adult beetles treated with LC50 of B. bassiana and spinetoram. Furthermore, the transcriptional response of Cytochrome P450 gene to each of the used insecticides in T. castaneum was examined by qRT-PCR. The results revealed that CYP450 was up-regulated by all the four insecticides tested. The highest level of up-regulation was recorded in adult beetles treated with LC50 of teflubenzuron (25.9-fold) followed by B. thuringienisis treated adults (7.6- fold), and spinetoram (6.8-fold). The least level of up-regulation was recorded in adults exposed to LC50 of B. bassiana (2.9- fold) as compared to control group.

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Screening of aptamers and their potential application in targeted diagnosis and therapy of liver cancer.

Aptamers are a class of single oligonucleotide molecules (DNA or RNA) that are screened from random DNA or RNA oligonucleotide chain libraries by the systemic evolution of ligands by exponential enrichment technology. The selected aptamers are capable of specifically binding to different targeting molecules, which is achieved by the three-dimensional structure of aptamers. Aptamers are similar in function to monoclonal antibodies, and therefore, they are also referred to as "chemical antibodies". Due to their high affinity and specificity and low immunogenicity, aptamers are topics of intense interest in today's biological targeting research especially in tumor research. They not only have high potential for clinical advances in tumor targeting detection but also are highly promising as targeted tumor drug carriers for use in tumor therapy. Various experimental studies have shown that aptamer-based diagnostic and therapeutic methods for liver cancer have great potential for application. This paper summarizes the structure, characteristics, and screening methods of aptamers and reviews the recent research progress on nucleic acid aptamers in the targeted diagnosis and treatment of liver cancer.

One-Pot Depolymerization and Polycondensation of PET Based Random Oligo- and Polyesters

The glycolysis of PET is a promising well-developed technique that could be utilized in a wide range of processes. Due to its equilibrial nature, the high ratio of reagents and the purification steps it is an expensive process. In this study our goal was to modify the standard glycolysis to an easily modulateable method (acido-alcoholysis) suited for one-pot oligoester, polyester synthesis. Adipic acid was added to the depolymerization reaction to connect low molecular weighed components into oligoesters. PET depolymerization reaction series were carried out with ethylene–glycol/adipic acid and butane-1,4-diol/adipic acid to demonstrate the tuneability of these systems. The reaction products were thoroughly characterized with NMR, FTIR, TGA, DSC and functional group analysis. One-pot depolymerization-polycondensation reactions were engineered to produce unsaturated and saturated polyesters. The unsaturated polyesters were dissolved in styrene, cross-linked and moulded to be characterized with DMA. A decreasing tendency of the glass transition temperature and cross-link density were found in the function of the ratio of adipic acid used during the depolymerization step. Acido-alcoholysis proved to be a versatile technique that could be used to engineer oligoesters and polyesters. The reagent use can be minimalized, and the raw reaction products can be directly applied as mid-products.

Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers.

This protocol describes the generation of radiolabeled cDNA probes from poly(A)+ RNA in a random priming reaction. Probes of this type are used for differential screening of cDNA libraries.

Screening of Oligonucleotide Aptamers and Application in Detection of Pesticide and Veterinary Drug Residues

Aptamers are a type of single stranded short deoxyribonucleic acid or ribonucleic acid sequences screened from a random oligonucleotide library, which have high affinity and specific recognition for specific target molecules. Since the advent of screening process called the systematic evolution of ligands by exponential enrichment (SELEX), there have been various SELEX-based improved screening processes and capillary electrophoresis-based SELEX or non-SELEX selection processes. As a molecular recognition element similar to antibody, aptamers have been applied to the detection of residues of pesticide and veterinary drugs associated with food and environmental safety. In these applications, aptamers are usually formed composite probes with other materials to produce signals, such as gold nanoparticles, quantum dots, or constitute sensors with electrochemical electrode. These screening methods and the applications of aptamers in the detection of pesticide and veterinary drug residues are summarized to provide a helpful reference for these issues.

Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides.

In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-32P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of Escherichia coli DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose.