Development of a DNA aptamer for direct and selective homocysteine detection in human serum

l-Homocysteine has been an amino acid intermediate of interest for over 20 years due to its implication in various adverse health conditions, including cardiovascular disease. Here, we report the first in vitro selection and application of high affinity aptamers for the target l-homocysteine. Two novel aptamer sequences were selected following 8 rounds of selection that displayed high affinity binding and selectivity to homocysteine compared to other amino acids. One of the selected aptamers, Hcy 8 (KD = 600 ± 300 nM), was used to develop a gold-nanoparticle biosensor capable of sensitive and selective homocysteine detection in human serum, with a limit of detection of 0.5 μM and a linear range of 0.5-3.0 μM. This biosensor allows rapid detection of free homocysteine in human serum samples at low cost, with little preparation time and could be adapted to be part of a po

Aptamer base: a collaborative knowledge base to describe aptamers and SELEX experiments

Over the past several decades, rapid developments in both molecular and information technology have collectively increased our ability to understand molecular recognition. One emerging area of interest in molecular recognition research includes the isolation of aptamers. Aptamers are single-stranded nucleic acid or amino acid polymers that recognize and bind to targets with high affinity and selectivity. While research has focused on collecting aptamers and their interactions, most of the information regarding experimental methods remains in the unstructured and textual format of peer reviewed publications. To address this, we present the Aptamer Base, a database that provides detailed, structured information about the experimental conditions under which aptamers were selected and their binding affinity quantified. The open collaborative nature of the Aptamer Base provides the community with a unique resource that can be updated and curated in a decentralized manner, thereby accommodating the ever evolving field of aptamer research

Amicus curiae on the request for consultative opinion formulated by the American Commission on Human Rights about Differentiated Approaches Regarding Persons Deprived of Liberty

These are reasons for amicus curiae presented to the Inter-American Court of Human Rights by the Center for Studies in Human Rights Systems and the Clinic for Access to Justice and Education in the Prisons of the Federal University of Paraná, on the occasion of the Request for Advisory Opinion (OC) number 29 formulated by the American Commission of Humans to the Inter-American Court, on different approaches to the matter of persons deprived of their liberty. The play is part of the context of the pandemic of COVID-19, which heightened vulnerabilities and subjected the prison population as a whole to a framework of hyper vulnerability, given the sanitary measures that imply reduced rights and weakened means of enforcement. Still, it advances on other contexts of overlapping vulnerabilities, such as concerning transgender women, the LGBTQI + public, pregnant women, breastfeeding women, and those accompanied by children in prison. Finally, the absence of the OC about other vulnerabilities not explicitly listed by the request of the IACHR, such as persons with disabilities deprived of liberty, with special emphasis on visual impairments and cases of need for use of wheelchairs with special needs, should be addressed.Trata-se de razões de amicus curiae apresentadas à Corte Interamericana de Direitos Humanos pelo Núcleo de Estudos em Sistemas de Direitos Humanos e Clínica de Acesso à Justiça e Educação nas Prisões da Universidade Federal do Paraná, por ocasião da Solicitação de Opinião Consultiva (OC) número 29 formulada pela Comissão Americana de Humanos à Corte Interamericana, sobre enfoques diferenciados em matéria de pessoas privadas de liberdade. A peça parte do contexto da pandemia da COVID-19 que aguçou vulnerabilidades e sujeitou a população carcerária como um todo a um quadro de hipervulnerabilidade, diante das medidas sanitárias que implicam em redução de direitos e em fragilização dos meios de fiscalização. Ainda, avança sobre outros contextos de vulnerabilidades superpostas como em relação às mulheres transgênero, o público LGBTQI+, mulheres gestantes, lactantes e acompanhadas de crianças no cárcere. Por fim, ataca-se as ausências da solicitação de OC em relação à outras vulnerabilidades não elencadas explicitamente pela solicitação da CIDH, como é o caso de pessoas com deficiência privadas de liberdade, com especial ênfase à deficiência visual e aos casos de necessidade de uso de cadeiras de rodas que necessitam de cuidados específicos e inexistentes no sistema penitenciário

Analysis of In Vitro Aptamer Selection Parameters

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success

Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology

<div><p>DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.</p></div

Aptasensor results.

<p>(A) Cyclic voltammograms of the norovirus aptasensor after each immobilization or binding step. The [Fe(CN)₆]^3−/2− redox couple was monitored for these experiments and cyclic voltammograms were recorded at a scan rate of 100 mV s^,–1 where (<i>a</i>) bare SPGE; (<i>b</i>) after self-assembly of the thiolated norovirus specific aptamer; (<i>c)</i> after back-filling with 1 mM 2-mercaptoethanol. (B) Square wave voltammograms obtained using (<i>a</i>) 20 aM, (<i>b</i>) 40 aM, (<i>c</i>) 60 aM, (<i>d</i>) 80 aM, (<i>e</i>) 100 aM, and (<i>f</i>) 120 aM of norovirus in buffer. (C) Calibration plot of current <i>vs.</i> concentration of norovirus. (D) Selectivity experiments performed using (a) buffer alones, (b) 5000 PFU of vesicular stomatitis virus, (c) 5.1 mg mL⁻¹ HSA, (d) 5000 PFU of vaccinia virus (e) 120 aM of norovirus. All experiments were performed in Dulbecco’s phosphate buffered saline after incubation with the developed aptasensor for 1 hr at 25°C. Square wave voltammograms were carried out in the range of −400 to 800 mV with a step potential of 4 mV, amplitude of 5 mV and frequency of 10 Hz. Electrochemical measurements were performed in 25 mM phosphate buffer (pH 7), containing 4 mM K₃[Fe(CN)₆] and 10 µM hexaamine ruthenium chloride.</p

Summary of aptamer candidates.

<p>Summary of aptamer candidates.</p