Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Stoltenburg, Regina; Strehlitz, Beate

doi:10.3390/ijms19020642

Cited by 19 publications

(17 citation statements)

References 51 publications

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“…To reliably bind a target molecule, the aptamer needs to adopt a stable 3D structure. Only with a stable structure the ligand can be bound efficiently by means of specific interactions [ 22 ]. Ligand-specific aptamers can be extracted by the established in vitro systematic evolution of ligands by exponential enrichment process (SELEX) [ 15 , 23 ].…”

Section: Introductionmentioning

confidence: 99%

Detailed Analysis of 17β-Estradiol-Aptamer Interactions: A Molecular Dynamics Simulation Study

Eisold

Labudde

2018

Molecules

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Micro-pollutants such as 17β-Estradiol (E2) have been detected in different water resources and their negative effects on the environment and organisms have been observed. Aptamers are established as a possible detection tool, but the underlying ligand binding is largely unexplored. In this study, a previously described 35-mer E2-specific aptamer was used to analyse the binding characteristics between E2 and the aptamer with a MD simulation in an aqueous medium. Because there is no 3D structure information available for this aptamer, it was modeled using coarse-grained modeling method. The E2 ligand was positioned inside a potential binding area of the predicted aptamer structure, the complex was used for an 25 ns MD simulation, and the interactions were examined for each time step. We identified E2-specific bases within the interior loop of the aptamer and also demonstrated the influence of frequently underestimated water-mediated hydrogen bonds. The study contributes to the understanding of the behavior of ligands binding with aptamer structure in an aqueous solution. The developed workflow allows generating and examining further appealing ligand-aptamer complexes.

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Section: Introductionmentioning

confidence: 99%

Detailed Analysis of 17β-Estradiol-Aptamer Interactions: A Molecular Dynamics Simulation Study

Eisold

Labudde

2018

Molecules

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“…In contrast, the survey of a non-exhaustive list of the sequences present in the R6 aptamer pool revealed eight different sequences out of 9 clones, but none similar to R10C1 and R10C5. This illustrates the directional evolution of the pool as a result of the additional positive selection rounds and counter-selection steps 48,49 . A strong bottleneck effect was likely caused by the last four positive selection rounds and the apparently stringent counter-selection rounds, which most likely led to the removal of several aptamers.…”

Section: Resultsmentioning

confidence: 99%

Identification of two aptamers binding toLegionella pneumophilawith high affinity and specificity

Saad

Chinerman

Tabrizian

et al. 2019

Preprint

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19Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires' Disease (LD) in 20 humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. 21 The methods currently available require expert skills and are time intensive, thus delaying 22 intervention. In situ detection of Lp by biosensor would allow rapid implementation of control 23 strategies. To this end, a biorecognition element is required. Aptamers are considered promising 24 biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding 25 into a specific structure and are able to bind to specific molecules. Currently no aptamer and thus 26 no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of 27 Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding 28 specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against 29 two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified 30 with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by 31 flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising 32 biorecognition molecules for the detection of Lp in water systems. 33 34 Key words: Legionella pneumophila, aptamers, SELEX, flow cytometry, fluorescence 35 microscopy, Pseudomonas 36 37 38 Legionella pneumophila (Lp) is a pathogenic Gram-negative bacterium responsible for two types 39 of respiratory diseases, namely the severe pneumonia Legionnaires' Disease (LD) and the milder 40 flu-like Pontiac fever 1 . Lp occurs in both natural and engineered water systems and is one of the 41 most prevalent pathogens in man-made, engineered water systems 2 . Infections occur when the 42 bacteria are aerosolized, and the contaminated aerosols are inhaled; Lp can then infect and replicate 43 inside alveolar macrophages 3 . Modern water systems provide optimal transmission conditions for 44 Lp by generating aerosols 4 . Leading sources of infection are cooling towers, hot water distribution 45 systems, humidifiers, misters, showers, fountains, spa pools and evaporative condensers 5 . 46 47 Outbreaks of LD consistently occur globally and have increased in recent years. The average 48 incidence rate is about 10-15 cases per million people 6 . According to the Centre for Disease 49 Control, incidences of legionellosis have increased by four and a half times between 2000 and 50 2016 7 . The rise in LD outbreaks can be attributed to several factors such as aging infrastructures 51and an aging population who is more vulnerable to such infections, as well as increases in diagnosis 52 and reporting 8,9 . Nevertheless, most LD outbreaks are the consequences of management failure of 53 man-made water systems 10 . Examples of these failures for water distribution systems include 54 keeping the temperature of hot water distribution system below 50°C and allowing water to 55 stagnate 10 . For coolin...

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“…The lengths of observed sequences obtained using NGS vary owing to insertions and/or deletions during the SELEX process. Stoltenburg and Strehiltz described that around 78% of sequences had an expected length of random regions, while the other 22% of sequences are different from the original length of random region [21]. Therefore, the probability P s,L was adjusted for different lengths of sequences using the following equation:…”

Section: String Score Definitionmentioning

confidence: 99%

FSBC: fast string-based clustering for HT-SELEX data

Kato

Ono

Minagawa³

et al. 2020

BMC Bioinformatics

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Background: The combination of systematic evolution of ligands by exponential enrichment (SELEX) and deep sequencing is termed high-throughput (HT)-SELEX, which enables searching aptamer candidates from a massive amount of oligonucleotide sequences. A clustering method is an important procedure to identify sequence groups including aptamer candidates for evaluation with experimental analysis. In general, aptamer includes a specific target binding region, which is necessary for binding to the target molecules. The length of the target binding region varies depending on the target molecules and/or binding styles. Currently available clustering methods for HT-SELEX only estimate clusters based on the similarity of full-length sequences or limited length of motifs as target binding regions. Hence, a clustering method considering the target binding region with different lengths is required. Moreover, to handle such huge data and to save sequencing cost, a clustering method with fast calculation from a single round of HT-SELEX data, not multiple rounds, is also preferred. Results: We developed fast string-based clustering (FSBC) for HT-SELEX data. FSBC was designed to estimate clusters by searching various lengths of over-represented strings as target binding regions. FSBC was also designed for fast calculation with search space reduction from a single round, typically the final round, of HT-SELEX data considering imbalanced nucleobases of the aptamer selection process. The calculation time and clustering accuracy of FSBC were compared with those of four conventional clustering methods, FASTAptamer, AptaCluster, APTANI, and AptaTRACE, using HT-SELEX data (>15 million oligonucleotide sequences). FSBC, AptaCluster, and AptaTRACE could complete the clustering for all sequence data, and FSBC and AptaTRACE performed higher clustering accuracy. FSBC showed the highest clustering accuracy and had the second fastest calculation speed among all methods compared. Conclusion: FSBC is applicable to a large HT-SELEX dataset, which can facilitate the accurate identification of groups including aptamer candidates. Availability of data and materials: FSBC is available at http://www.aoki.ecei.tohoku.ac.jp/fsbc/.

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Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Cited by 19 publications

References 51 publications

Detailed Analysis of 17β-Estradiol-Aptamer Interactions: A Molecular Dynamics Simulation Study

Detailed Analysis of 17β-Estradiol-Aptamer Interactions: A Molecular Dynamics Simulation Study

Identification of two aptamers binding toLegionella pneumophilawith high affinity and specificity

FSBC: fast string-based clustering for HT-SELEX data

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