703 research outputs found

    Aptamer-based therapeutics and their potential in radiopharmaceutical design

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    Aptamers, short, single stranded oligonucleotide entities, have been developed in the past 15 years against a plethora of targets and for a variety of applications. These range from inhibition of receptors and enzymes to the identification of small molecules in sensor applications, and from the development of targeted therapeutic to the design of novel diagnostic and imaging agents. Furthermore, aptamers have been designed for targets that cover a wide range of diseases, from HIV to tropical diseases, cancer and inflammation. Their easy development and flexibility of use and manipulation, offers further potential. In this paper we review their selection and consider some of the recent applications of aptamers in the design of radiopharmaceuticals for the targeted radiotherapy and medical imaging of disease

    Dna Aptamers Selected Against Wild-Type Helix 69 Ribosomal Rna And Their Implications In Combating Antibiotic Resistance

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    Outbreaks of advanced antibiotic-resistant strains of microbes have hastened the need to identify new viable molecular targets for the development of novel anti-infectives. For this purpose, helix 69 (H69, or m3a 19-nucleotide (nt) hairpin motif that is highly conserved throughout phylogeny and rich in modified nucleotides, including pseudouridine () and 3-methylpseudouridine (m3) was chosen as a potential target. Helix 69, which is located in domain IV of Escherichia coli 23S ribosomal RNA (rRNA), undergoes conformational changes when in close proximity to the decoding region of 16S rRNA and transfer RNAs (tRNAs) in the peptidyl-transferase center (PTC). Functionally, the exact biological role(s) of H69 remains unclear; however, its proposed importance within protein synthesis may support it as an ideal target to develop ligands with high binding specificity. In this thesis work, DNA aptamer candidates with binding specificity for the wild-type bacterial H69 were selected. The 84-nt-long DNA aptamer (H69DNAapt18) that was identified from a DNA library with a 40-nt randomized region has the sequence 5\u27-CTCCCCGGGCACTATTTCCTGGGACTAGTTCTGCAGGTTT-3\u27. The initial library contained 5 Ă— 1014 DNA sequences and was used in SELEX (systematic evolution of ligands by exponential enrichment) (Molecular diversity of the library was reduced to approximately 1 102 after 11 rounds of in vitro selection) experiments. A synthetic construct of H69 was biotinylated and used with optimized SELEX. After immobilization of the biotinylated target H69 to streptavidin-coated surfaces, DNA library candidates were challenged against H69 in multiple rounds of selection, recovered, and enriched by direct-bead PCR (polymerase chain reaction). Levels of bound DNA and diversity of the amplified library pools were monitored by UV-visible spectroscopy and sequencing between rounds of SELEX (11 total). Select rounds were cloned and sequenced. Consensus sequences from select rounds of SELEX were identified by using Clustal W alignments, and optimal secondary structures were predicted by Mfold analysis. Analysis of 120 clones led to the identification of 20 sequences with consensus motifs. Notably, one of the selected DNA ligands (H69DNAapt18) contained a conserved 20-nt hairpin-loop motif with complementarity to the loop region of the targeted E. coli wild-type H69. Interestingly, this 20-nt hairpin motif of H69DNAapt18 retained its conserved 20-nt motif within the truncated 40-nt Mfold structural prediction representing only the randomized region of the 84-nt DNA library. Attempts to determine binding affinities of select isolated DNA aptamer candidates to 32P-radiolabeled H69 by electrophoretic mobility shift assays (EMSAs) were moderately successful, with observation of RNA-DNA complexes with apparent dissociation constants (Kds) in the high nM range. To better evaluate the affinity and selectivity of DNA aptamer #18, a fluorescently tagged H69-DNA aptamer #18 (FAM-H69DNAapt18) was used for a comparison binding study with wild-type H69 and unmodified rRNA constructs. Relative dissociation constants of FAM-H69DNAapt18 for H69 and RNAs other than H69, as determined by fluorescence titrations with small RNA constructs, are as follows: wild-type H69 \u3e partially modified H69 \u3e unmodified H69 \u3e A-site RNA of 16S rRNA. Overall, this study provides a reference point for the development of DNA aptamers that identify modified nucleotides and/or methylation sites in RNA, or could potentially function as novel therapeutics to help combat antibiotic resistance. Keywords: 23S ribosomal RNA (rRNA); helix 69 (H69); modified nucleotides; pseudouridine (); 3-methylpseudouridine (m3); peptidyl-transferase center (PTC); systematic evolution of ligands by exponential enrichment (SELEX); aptamers; fluorescence spectroscopy; DNA:RNA complexes; dissociation constant (Kd); anti-infectives; antibiotic resistance; antibacterial therapeutics; and bioprobes

    The identification of aptamers against serum biomarkers of human tuberculosis

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    >Magister Scientiae - MScTuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment

    From Tag to Target: Development of Strategies to avoid Selection of His-tag-binding Aptamers using the Example of N-terminally His-tagged Carbapenemases KPC-2 and NDM-1 for the Implementation of Carbapenemase-specific Aptamers in Lateral Flow Devices

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    The rapid increase in occurrence of carbapenemase producing multi drug resistant Entero-bacteriaceae represents a world wide health risk. Carbapenem antibiotics have long been used as a treatment of last resort for infections with multi resistant bacteria. Now that bacteria develop enzymes that render these agents useless, treatment options are limited, and the health care system is left with a fast-growing challenge. Easy to use and cost-effective detection of carbapenem resistance and discrimination between different carbapenemases are important to prevent spread and to facilitate appropriate treatment. The development of reliable detection systems strongly depends on the availability of highly affine and specific molecular recognition molecules. Aptamers are promising molecular recognition molecules. They are small and chemically synthesized, single stranded (ss) DNA or RNA molecules that bind to their targets with high affinity and specificity. Aptamers are selected by an in vitro selection process that can be tailored to suit the desired application. Chemical synthesis of aptamers minimizes batch-to-batch variations and facilitates easy and cost-effective modifications. Yet, an effective aptamer selection process is often hampered by non-specific binding to side targets. Thus, negative selections against potential side targets are recommended. In this work, selections were carried out against N-terminally polyhistidine (His)-tagged New Delhi metallo-beta-lactamase 1 (His-NDM-1) and Klebsiella pneumoniae carbapenemase-2 (His-KPC-2). Eleven selection rounds (SRs), partially conducted with negative selections against His-tagged carbapenemases did not result in the identification of aptamers. Three consecutive SRs without negative selections against His-tagged proteins resulted in the identification of high-affinity His-tag aptamers, only one His-KPC-2 and no His-NDM-1 aptamer. To facilitate carbapenemase binding while preventing His-tag binding, the selection against His-KPC-2 was restarted from SR eleven and three different selection strategies were designed. The strategies included masking of the His-tag and competitive elution of His-tag-binding sequences using a truncated version of the previously selected His-tag aptamer as well as immobilization of the protein via its His-tag. After three SRs, sequencing data was analyzed for the enrichment of a motif that is likely involved in His-tag binding. Based on this analysis, the masking approach was identified as the most promising strategy. Binding to His-KPC-2 was demonstrated for several aptamers. Two of these aptamers were further characterized for binding properties. Both very likely did not bind to the His-tag. Consequently, an aptamer-based lateral flow assay was developed for His-KPC-2. The masking approach was also applied to selection against His-NDM-1. Here, an aptamer with a binding affinity in the high nanomolar to low micromolar range, without cross reactivity to another His-tagged carbapenemase and a synthetic hexa-His peptide was identified. This aptamer may serve as a valuable tool for the detection of the very prevalent and harmful carbapenemase NDM-1

    Prostate-specific Rna Aptamer: Promising Nucleic Acid Antibody-like Cancer Detection

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    Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.5Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2012/24911-0]CNPq [490574/2010-6, 457420/2013-8

    RNA Aptamer Evolution: Two Decades of SELEction

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    Aptamers are small non-coding RNAs capable of recognizing, with high specificity and affinity, a wide variety of molecules in a manner that resembles antibodies. This class of nucleic acids is the resulting product of applying a well-established screening method known as SELEX. First developed in 1990, the SELEX process has become a powerful tool to select structured oligonucleotides for the recognition of targets, starting with small molecules, going through protein complexes until whole cells. SELEX has also evolved along with new technologies positioning itself as an alternative in the design of a new class of therapeutic agents in modern molecular medicine. This review is an historical follow-up of SELEX method over the two decades since its first appearance

    Function and dynamics of aptamers: A case study on the malachite green aptamer

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    Aptamers are short single-stranded nucleic acids that can bind to their targets with high specificity and high affinity. To study aptamer function and dynamics, the malachite green aptamer was chosen as a model. Malachite green (MG) bleaching, in which an OH- attacks the central carbon (C1) of MG, was inhibited in the presence of the malachite green aptamer (MGA). The inhibition of MG bleaching by MGA could be reversed by an antisense oligonucleotide (AS) complementary to the MGA binding pocket. Computational cavity analysis of the NMR structure of the MGA-MG complex predicted that the OH- is sterically excluded from the C1 of MG. The prediction was confirmed experimentally using variants of the MGA with changes in the MG binding pocket. This work shows that molecular reactivity can be reversibly regulated by an aptamer-AS pair based on steric hindrance. In addition to demonstrate that aptamers could control molecular reactivity, aptamer dynamics was studied with a strategy combining molecular dynamics (MD) simulation and experimental verification. MD simulation predicted that the MG binding pocket of the MGA is largely pre-organized and that binding of MG involves reorganization of the pocket and a simultaneous twisting of the MGA terminal stems around the pocket. MD simulation also provided a 3D-structure model of unoccupied MGA that has not yet been obtained by biophysical measurements. These predictions were consistent with biochemical and biophysical measurements of the MGA-MG interaction including RNase I footprinting, melting curves, thermodynamic and kinetic constants measurement. This work shows that MD simulation can be used to extend our understanding of the dynamics of aptamer-target interaction which is not evident from static 3D-structures. To conclude, I have developed a novel concept to control molecular reactivity by an aptamer based on steric protection and a strategy to study the dynamics of aptamer-target interaction by combining MD simulation and experimental verification. The former has potential application in controlling metabolic reactions and protein modifications by small reactants and the latter may serve as a general approach to study the dynamics of aptamer-target interaction for new insights into mechanisms of aptamer-target recognition
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