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Bio-Inspired DNA Nanoswitches and Nanomachines: Applications in Biosensing and Drug Delivery
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Arnaud Desrosiers, Alexis Vallée-Bélisle
In the event that a specific input target of interest has no known DNA binding sequence, SELEX methods can be employed to identify nucleotide sequences that display high affinity for small organics [92], proteins [93,94] and even viruses [95] or whole bacteria [96] (mostly through surface proteins/sugars). Briefly, SELEX (short for systematic evolution of ligands by exponential enrichment) is a method developed in 1990 [37,97] in which specific nucleotide sequences (referred to as aptamer with typically 15–40 nucleotides) from random libraries are selected to bind specific targets with high affinity through several rounds of selection. This method was first developed for RNA selection but was then rapidly adapted to select DNA sequences [36]. In this method, one selection round typically consists in a simple binding experiment where DNA sequences bound to the target of interest are amplified and used for the next selection round.
From RNA Structures to RNA Nanomachines
Published in Yubing Xie, The Nanobiotechnology Handbook, 2012
Sabarinath Jayaseelan, Paul D. Kutscha, Francis Doyle, Scott A. Tenenbaum
There are numerous examples of RNA binding to small ligands from organic molecules such as amino acids, guanosine, or cofactors to large dye molecules such as ethidium bromide or SYBR gold, which are used as RNA-imaging agents (Chow and Bogdan 1997). Creating a type of artificial hybrid association with RNA is like creating a key to a constantly changing dynamic lock. This is where powerful techniques like SELEX (systematic evolution of ligands by exponential enrichment) (Tuerk and Gold 1990) have proven useful. In SELEX, a large library of the intended molecules, typically oligonucleotides, is selected against a target (e.g., protein) over several rounds of positive and negative selection. This technique can be used for creating hybrid functional networks and not just for elaborate RNA architecture production.
Aptamers and Cancer Nanotechnology
Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006
Omid C. Farokhzad, Sangyong Jon, Robert Langer
In vitro selection,25 also called systematic evolution of ligands by exponential enrichment(SELEX),26,48 is a protocol to isolate rare functional oligonucleotides (i.e., aptamers) from a pool of random oligonucleotides (Figure 16.1). Similar to phage display or other strategies used to isolate ligands from random libraries, SELEX is essentially an iterative selection and amplification protocol to isolate single-stranded nucleic acid ligands that bind to their target with high affinity and specificity. The complexity of the starting library is determined in part by the number of random nucleotides in the pool. For example, by using a library with 40 random nucleotides, a pool of 1024 distinct nucleotides can be generated. Practically speaking, the number of ligands in the starting pool for in vitro selection is closer to 1015, representing 1 nmol of the library.
Aptamer based tools for environmental and therapeutic monitoring: A review of developments, applications, future perspectives
Published in Critical Reviews in Environmental Science and Technology, 2020
Błażej Kudłak, Monika Wieczerzak
SELEX in vitro selection is the most widely used method for obtaining RNA and single strand DNA (ssDNA) aptamers with the desired physical and chemical properties. In the first step, a very large library of oligonucleotides is synthesized, consisting of randomly generated sequences and shapes of constant length surrounded by 5′ and 3′ ends. During the synthesis of different regions, monomer concentrations (pyridine and purine bases A, T/U, G, and C) in the reaction mixture are equimolar, so the probability of occurrence for each oligonucleotide combination is the same. In the case of n-nucleotide fragments, this gives 4n different combinations; a pool of oligonucleotides that theoretically represent every possible nucleotide sequence (Groher & Suess, 2016).
Aptamer-based technology for radionuclide targeted imaging and therapy: a promising weapon against cancer
Published in Expert Review of Medical Devices, 2020
Luca Filippi, Oreste Bagni, Clara Nervi
The standard process for producing DNA and RNA aptamers is represented by the already cited SELEX method [11]. In brief, SELEX requires the molecular target of interest to be incubated with a pool of 1014 ~ 1016 single-stranded nucleic acids (DNA or RNA libraries), harboring random sequences of 20–60 bases flanked, at the 5ʹ and 3ʹ ends, by fixed regions including primer sites for polymerase chain reaction (PCR) amplification. High-affinity target-bound oligonucleotides are enriched from library species population by repetitive rounds of selection, consisting of partitioning unbound sequences in the library from target-bound sequences, elution, and amplification of the nucleic acid pool through PCR. A specific selection stringency of the enriched aptamers is obtained by varying experimental binding conditions (e.g. ion strength, pH temperature, pI) affecting the affinity and function of the enriched aptamers. The main differences between SELEX protocols for the identification of DNA and RNA aptamers are the need to protect RNA from degradation induced by RNAases, amplification through RNA polymerase and, most importantly, the need of reverse transcription before PCR. Since tridimensional folding is of crucial importance in determining the affinity between an aptamer and its target, immunoprecipitation-coupled SELEX (IP-SELEX) has been developed to plunge the molecular target in its native 3D conformation through immunoprecipitation, thus improving the selection and enrichment of aptamers with high affinity for proteins in their native configuration. However, further methods have been introduced for improving the original SELEX approach, among whom cell-SELEX is worthy of note for its potential applications in oncology [12]. Cell-SELEX has been developed with the aim of producing aptamers specifically directed toward a cell of interest, usually a cancer cell, by incubating a library of DNA oligonucleotides with the target cell [13]. In such a way, a variety of aptamers with specific affinity for cell lines derived from many cancers (i.e. breast, prostate, and ovarian cancers) have been obtained [14].