Generating aptamers towards human sperm cells using massively parallel sequencing

Gooch, James; Tungsirisurp, Sireethorn; Costanzo, Hayley; Napier, Richard; Frascione, Nunzianda

doi:10.1007/s00216-021-03562-7

Cited by 9 publications

(5 citation statements)

References 34 publications

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“…The sperm cell isolation was carried out using density gradient centrifugation following the 5th WHO standard. Percoll (Sigma-Aldrich, Dorset, UK) and ×10 Human Tubal Fluid (HTF) were used to prepare an iso-osmotic gradient [ 23 ]. Briefly, discontinuous double-density gradients with two layers (2 mL 40% and 2 mL 80%) were prepared.…”

Section: Methodsmentioning

confidence: 99%

DNA methylation patterns in patients with asthenospermia and oligoasthenospermia

Zhang,

Li,

Wang

et al. 2024

BMC Genomics

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Background Spermatogenesis is a highly regulated and complex process in which DNA methylation plays a crucial role. This study aimed to explore the differential methylation profiles in sperm DNA between patients with asthenospermia (AS) and healthy controls (HCs), those with oligoasthenospermia (OAS) and HCs, and patients with AS and those with OAS. Results Semen samples and clinical data were collected from five patients with AS, five patients with OAS, and six age-matched HCs. Reduced representation bisulfite sequencing (RRBS) was performed to identify differentially methylated regions (DMRs) in sperm cells among the different types of patients and HCs. A total of 6520, 28,019, and 16,432 DMRs were detected between AS and HC, OAS and HC, and AS and OAS groups, respectively. These DMRs were predominantly located within gene bodies and mapped to 2868, 9296, and 9090 genes in the respective groups. Of note, 12, 9, and 8 DMRs in each group were closely associated with spermatogenesis and male infertility. Furthermore, BDNF, SMARCB1, PIK3CA, and DDX27; RBMX and SPATA17; ASZ1, CDH1, and CHDH were identified as strong differentially methylated candidate genes in each group, respectively. Meanwhile, the GO analysis of DMR-associated genes in the AS vs. HC groups revealed that protein binding, cytoplasm, and transcription (DNA-templated) were the most enriched terms in the biological process (BP), cellular component (CC), and molecular function (MF), respectively. Likewise, in both the OAS vs. HC and AS vs. OAS groups, GO analysis revealed protein binding, nucleus, and transcription (DNA-templated) as the most enriched terms in BP, CC, and MF, respectively. Finally, the KEGG analysis of DMR-annotated genes and these genes at promoters suggested that metabolic pathways were the most significantly associated across all three groups. Conclusions The current study results revealed distinctive sperm DNA methylation patterns in the AS vs. HC and OAS vs. HC groups, particularly between patients with AS and those with OAS. The identification of key genes associated with spermatogenesis and male infertility in addition to the differentially enriched metabolic pathways may contribute to uncovering the potential pathogenesis in different types of abnormal sperm parameters.

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Section: Methodsmentioning

confidence: 99%

DNA methylation patterns in patients with asthenospermia and oligoasthenospermia

Zhang,

Li,

Wang

et al. 2024

BMC Genomics

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“…Firstly, Galaxy was used with an automated, custom Cell-SELEX pipeline that is capable of processing FASTQ files (both forward and reverse read files) that were generated as a result of MPS. Stepwise information for this custom pipeline has been reported in 2021 by Gooch et al [25]. In order to validate results obtained from the Galaxy workflow, AptaSUITE was employed in order to gain further insight into features of the pools, such as base distribution and enrichment.…”

Section: Massively Parallel Sequencing and Data Processingmentioning

confidence: 99%

“…The round 6, 8 and 10 library pools were then loaded into a V2 reagent cartridge and sequenced using the Illumina MiSeq instrument (San Diego, CA, USA). Data analysis of the FASTQ files generated by the Illumina MiSeq was conducted using Galaxy, an open-source bioinformatics platform, using a pipeline developed in house that was designed for use with Cell-SELEX library pools following MPS [23,25]. Further analysis was conducted using AptaSUITE, an aptamer-specific bioinformatics software designed for the analysis of MPS aptamer pool files [24].…”

Section: Massively Parallel Sequencingmentioning

confidence: 99%

“…Using two bioinformatic programmes, Galaxy and AptaSUITE, the MPS datasets were processed to elucidate binding sequences [23,24]. The Galaxy pipeline used within this project was developed in house and was adapted for use with the specific library for this SELEX selection [25]. Aptamer binding capability and selectivity were investigated using Quantitative Polymerase Chain Reaction (qPCR) and fluorescent microscopy.…”

Section: Introductionmentioning

confidence: 99%

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The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells

Costanzo,

Gooch,

Tungsirisurp

et al. 2024

IJMS

Self Cite

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Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive but not specific, so small amounts of the substrate can be detected, but false-positive results are often encountered, which can be misleading. Novel methods for the detection of red blood cells based on aptamer–target interactions may be able to overcome these issues. Aptamers are single-stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The use of aptamers as a target-specific moiety poses several advantages and has the potential to replace antibodies within immunoassays. Aptamers are cheaper to produce, display no batch-to-batch variation and can allow for a wide range of chemical modifications. They can help limit cross-reactivity, which is a hindrance to current forensic testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process was used to generate aptamers against whole red blood cells. Obtained aptamer pools were analysed via massively parallel sequencing to identify viable sequences that demonstrate a high affinity for the target. Using bioinformatics platforms, aptamer candidates were identified via their enrichment profiles. Binding characterisation was also conducted on two selected aptamer candidates via fluorescent microscopy and qPCR to visualise and quantify aptamer binding. The potential for these aptamers is broad as they can be utilised within a range of bioassays for not only forensic applications but also other analytical science and medical applications. Potential future work includes the incorporation of developed aptamers into a biosensing platform that can be used at crime scenes for the real-time detection of human blood.

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“…Aptamers are now widely used as biosensors in a large number of fields [291,292] with significant and recent examples seen for the rapid detection of SARS-CoV-2 [293,294] and have potential for the use in forensic science [295]. Aptamers have been developed to detect PSA [296], hemoglobin [297] and more recently, aptamers to detect spermatozoa have been described using a combination of Cell-SELEX and massively parallel sequencing technologies [295]. Once suitably specific and sensitive aptamers are obtained, they can be built into a range of sensors for detection many of them suitable for field deployment.…”

Section: Aptamersmentioning

confidence: 99%

On the Identification of Body Fluids and Tissues: A Crucial Link in the Investigation and Solution of Crime

Sijen

Harbison

2021

Genes

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Body fluid and body tissue identification are important in forensic science as they can provide key evidence in a criminal investigation and may assist the court in reaching conclusions. Establishing a link between identifying the fluid or tissue and the DNA profile adds further weight to this evidence. Many forensic laboratories retain techniques for the identification of biological fluids that have been widely used for some time. More recently, many different biomarkers and technologies have been proposed for identification of body fluids and tissues of forensic relevance some of which are now used in forensic casework. Here, we summarize the role of body fluid/ tissue identification in the evaluation of forensic evidence, describe how such evidence is detected at the crime scene and in the laboratory, elaborate different technologies available to do this, and reflect real life experiences. We explain how, by including this information, crucial links can be made to aid in the investigation and solution of crime.

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Generating aptamers towards human sperm cells using massively parallel sequencing

Cited by 9 publications

References 34 publications

DNA methylation patterns in patients with asthenospermia and oligoasthenospermia

DNA methylation patterns in patients with asthenospermia and oligoasthenospermia

The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells

On the Identification of Body Fluids and Tissues: A Crucial Link in the Investigation and Solution of Crime

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