RESUMO
Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodosRESUMO
The ability to quantify cocaine in biological fluids is crucial for both the diagnosis of intoxication and overdose in the clinic as well as investigation of the drug's pharmacological and toxicological effects in the laboratory. To this end, we have performed high-stringency in vitro selection to generate DNA aptamers that bind cocaine with nanomolar affinity and clinically relevant specificity, thus representing a dramatic improvement over the current-generation, micromolar-affinity, low-specificity cocaine aptamers. Using these novel aptamers, we then developed two sensors for cocaine detection. The first, an in vitro fluorescent sensor, successfully detects cocaine at clinically relevant levels in 50% human serum without responding significantly to other drugs of abuse, endogenous substances, or a diverse range of therapeutic agents. The second, an electrochemical aptamer-based sensor, supports the real-time, seconds-resolved measurement of cocaine concentrations in vivo in the circulation of live animals. We believe the aptamers and sensors developed here could prove valuable for both point-of-care and on-site clinical cocaine detection as well as fundamental studies of cocaine neuropharmacology.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Cocaína , Animais , Humanos , Aptâmeros de Nucleotídeos/química , Soro , Cocaína/químicaRESUMO
Synthetic cannabinoids are a widely abused class of dangerous psychoactive substances, especially among youths and young adults. Dozens of such drugs have been identified to date, and new ones continue to emerge. The ability to detect these drugs is important for interdiction efforts and the diagnosis of drug overdose, but existing analytical methods lack broad cross-reactivity to diverse members of this drug family. Here, we have utilized library-immobilized SELEX to generate DNA aptamers that can broadly recognize various members of the indazole-3-carboxamide synthetic cannabinoid family. Using two representatives of this family, AB-FUBINACA and 5F-AMB, we identify two aptamers FUB4 and AMB2F with respective dissociation constants (KDs) of 138 ± 15 and 411 ± 20 nM for their targets. These aptamers can recognize many indazole-based synthetic cannabinoids with high affinity and excellent specificity against natural cannabinoids as well as other structurally similar interferents like serotonin and tryptophan. We use these two aptamers to develop fluorescence strand-displacement sensors that successfully detect these synthetic cannabinoids at concentrations as low as 50 nM in human serum. The sensors can also detect up to 14 different drugs from this familyâa major improvement over the six recognized by an existing commercial immunoassay.
Assuntos
Aptâmeros de Nucleotídeos , Canabinoides , Indazóis , Aptâmeros de Nucleotídeos/química , Indazóis/química , Canabinoides/química , Técnica de Seleção de Aptâmeros , HumanosRESUMO
Conventional directed evolution methods offer the ability to select bioreceptors with high binding affinity for a specific target in terms of thermodynamic properties. However, there is a lack of analogous approaches for kinetic selection, which could yield affinity reagents that exhibit slow off-rates and thus remain tightly bound to targets for extended periods. Here, we describe an in vitro directed evolution methodology that uses the nuclease flap endonuclease 1 to achieve the efficient discovery of aptamers that have slow dissociation rates. Our nuclease-assisted selection strategy can yield specific aptamers for both small molecules and proteins with off-rates that are an order of magnitude slower relative to those obtained with conventional selection methods while still retaining excellent overall target affinity in terms of thermodynamics. This new methodology provides a generalizable approach for generating slow off-rate aptamers for diverse targets, which could, in turn, prove valuable for applications including molecular devices, bioimaging, and therapy.
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Cinética , TermodinâmicaRESUMO
Opioids collectively cause over 80,000 deaths in the United States annually. The ability to rapidly identify these compounds in seized drug samples on-site will be essential for curtailing trafficking and distribution. Chemical reagent-based tests are fast and simple but also notorious for giving false results due to poor specificity, whereas portable Raman spectrometers have excellent selectivity but often face interference challenges with impure drug samples. In this work, we develop on-site sensors for morphine and structurally related opioid compounds based on in vitro-selected oligonucleotide affinity reagents known as aptamers. We employ a parallel-and-serial selection strategy to isolate aptamers that recognize heroin, morphine, codeine, hydrocodone, and hydromorphone, along with a toggle-selection approach to isolate aptamers that bind oxycodone and oxymorphone. We then utilize a new high-throughput sequencing-based approach to examine aptamer growth patterns over the course of selection and a high-throughput exonuclease-based screening assay to identify optimal aptamer candidates. Finally, we use two high-performance aptamers with KD of â¼1 µM to develop colorimetric dye-displacement assays that can specifically detect opioids like heroin and oxycodone at concentrations as low as 0.5 µM with a linear range of 0-16 µM. Importantly, our assays can detect opioids in complex chemical matrices, including pharmaceutical tablets and drug mixtures; in contrast, the conventional Marquis test completely fails in this context. These aptamer-based colorimetric assays enable the naked-eye identification of specific opioids within seconds and will play an important role in combatting opioid abuse.
RESUMO
We reported over 20 years ago MNS-4.1, the first DNA aptamer with a micromolar affinity for cocaine. MNS-4.1 is based on a structural motif that is very common in any random pool of oligonucleotides, and it is actually a nonspecific hydrophobic receptor with wide cross-reactivity with alkaloids and steroids. Despite such weaknesses preventing broad applications, this aptamer became widely used in proof-of-concept demonstrations of new formats of biosensors. We now report a series of progressively improved DNA aptamers recognizing cocaine, with the final optimized receptors having low nanomolar affinity and over a thousand-fold selectivity over the initial cross-reactants. In the process of optimization, we tested different methods to eliminate cross-reactivities and improve affinity, eventually achieving properties that are comparable to those of the reported monoclonal antibody candidates for the therapy of overdose. Multiple aptamers that we now report share structural motifs with the previously reported receptor for serotonin. Further mutagenesis studies revealed a palindromic, highly adaptable, broadly cross-reactive hydrophobic motif that could be rebuilt through mutagenesis, expansion of linker regions, and selections into receptors with exceptional affinities and varying specificities.