ABSTRACT
The COVID-19 pandemic has been a main concern over the last two years and has become one of the most important crises in the history of human health. Today, there is still a need for affordable and reliable diagnostic tests for massive disease monitoring. Previously, a set of highly specific DNA-aptamers (C7/C9) binding to the SARS-CoV-2 Spike (S) protein were isolated but its performance in clinical samples remained to be tested. Here, 242 samples were collected through three different methods and subjected to florescence-linked aptamer assays (FLAA) based on C7/C9 aptamers through two readout protocols. Then, a step-by-step statistical approach which included agreement tests, proportion comparisons and binomial and multinomial logistic regressions was used to predict optimal conditions for the novel C7/C9 FLAA test. RTqPCR threshold cycles, symptoms onset and processing time were influential factors on FLAA test results. Naturally occurring mutations on S were also detected and analyzed. Aminoacidic substitutions D614G and T732A appeared relevant for aptamer recognition although further studies are necessary. The methodology presented here is the first step to determine the performance and diagnosis across a range of clinical contexts and it might serve as a base for a complete analysis applicable to other designs of new diagnostic tests.
ABSTRACT
The rapid spread of SARS-CoV-2 infection throughout the world led to a global public health and economic crisis triggering an urgent need for the development of low-cost vaccines, therapies and high-throughput detection assays. In this work, we used a combination of Ideal-Filter Capillary Electrophoresis SELEX (IFCE-SELEX), Next Generation Sequencing (NGS) and binding assays to isolate and validate single-stranded DNA aptamers that can specifically recognize the SARS-CoV-2 Spike glycoprotein. Two selected non-competing DNA aptamers, C7 and C9 were successfully used as sensitive and specific biological recognition elements for the development of electrochemical and fluorescent aptasensors for the SARS-CoV-2 Spike glycoprotein with detection limits of 0.07 fM and 41.87 nM, respectively.
Subject(s)
Aptamers, Nucleotide , COVID-19 , Aptamers, Nucleotide/genetics , COVID-19/diagnosis , Humans , SARS-CoV-2/genetics , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
The Let-7:LIN28 regulatory loop is a paradigm in miRNA regulation. LIN28 harbors two RNA binding domains, which interact with well-conserved sequences in pre-let-7 RNAs, the GNGAY and the GGAG motifs. Here, the differential binding between LIN28B and pre-let-7 members was associated with the structural characteristics of the pre-let-7 family mapped by SHAPE, uncovering diverse structural patterns within pre-let-7 members. Pre-let-7 mutants supported a relevant role of the GGAG motif location and the preE-stem stability for the interaction with LIN28B. Based on these results, we propose a core RNA structure for LIN28B interaction.
