Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

Aptamerer är nukleinsyra-baserade molekyler som binder specifikt till en målstruktur. Aptamerer har flera fördelar över antikroppar, så som snabb och billig framtagning och produktion. Multimera aptamer-baserade strukturer har visats ge bättre resultat än monomera aptamerer, men framtagningen av sådana strukturer är tidskrävande och icke-skalbar. Denna studie utforskar generering och selektion av multimera aptamer-baserade strukturer genom slumpmässig ligering och in vitro-evolution, med virusliknande partiklar av norovirus GII.2 och GII.4 som målstruktur. Optimering av ligeringsförhållanden visade att en större andel aptamerer, flerarmade fragment, och linjära fragment resulterade i störst diversitet i den ursprungliga strukturblandingen. Selektionsexperiment uppvisade kraftig positiv selektion för strukturer innehållande aptameren Buf-2, vilket indikerar att den har hög affinitet för båda genotyper. Aptameren SMV21 uppvisade också positiv selektion för båda genotyper. Studien finner också positiv selektion av multimera aptamer-baserade strukturer, vilket bekräftar att de binder starkare till sin målstruktur. Potentiella sekvenser med hög affinitet togs fram genom att generera konsensus-sekvenser från sekvenseringsdatan av de selekterade strukturerna. SPR användes för att mäta affiniteten av de selekterade strukturerna till norovirus, men på grund av ospecifik binding kunde inga slutsatser dras. / Aptamers are nucleic acid-based targeted binders that hold advantages over antibodies, such as cheap and fast development and production. Multimeric aptamer-based structures have shown improved performance compared to monomeric aptamers, but the development of such structures is time-consuming and unscalable. This study explores the generation and selection of multimeric aptamer-based structures through random ligation and in vitro evolution, targeting norovirus GII.2 and GII.4. Optimization of ligation conditions was performed, revealing that a bigger proportion of aptamers, multi-armed fragments, and linear fragments ensures a diverse initial structure pool. Selection experiments demonstrated a strong positive selection for structures containing the Buf-2 aptamer, indicating its high affinity for both norovirus genotypes. The SMV21 aptamer also showed positive selection for both genotypes. The study further found that multimeric aptamer-based structures experience positive selection, confirming their stronger binding to the desired target. Potential strong-binding sequences were obtained by generating consensus sequences from sequencing data of the selected strong binders. SPR was employed to determine the affinity of the selected binders to the norovirus, but the results were inconclusive due to unspecific binding.