Development of Better Aptamers: Structured Library Approaches, Selection Methods, and Chemical Modifications.

Systematic evolution of ligands by exponential enrichment (SELEX) has been used to discover thousands of aptamers since its development in 1990. Aptamers are short single-stranded oligonucleotides capable of binding to targets with high specificity and selectivity through structural recognition. While aptamers offer advantages over other molecular recognition elements such as their ease of production, smaller size, extended shelf-life, and lower immunogenicity, they have yet to show significant success in real-world applications. By analyzing the importance of structured library designs, reviewing different SELEX methodologies, and the effects of chemical modifications, we provide a comprehensive overview on the production of aptamers for applications in drug delivery systems, therapeutics, diagnostics, and molecular imaging.

An RNA-Cleaving DNAzyme That Requires an Organic Solvent to Function.

Engineering functional nucleic acids that are active under unusual conditions will not only reveal their hidden abilities but also lay the groundwork for pursuing them for unique applications. Although many DNAzymes have been derived to catalyze diverse chemical reactions in aqueous solutions, no prior study has been set up to purposely derive DNAzymes that require an organic solvent to function. Herein, we utilized in vitro selection to isolate RNA-cleaving DNAzymes from a random-sequence DNA pool that were "compelled" to accept 35 % dimethyl sulfoxide (DMSO) as a cosolvent, via counter selection in a purely aqueous solution followed by positive selection in the same solution containing 35 % DMSO. This experiment led to the discovery of a new DNAzyme that requires 35 % DMSO for its catalytic activity and exhibits drastically reduced activity without DMSO. This DNAzyme also requires divalent metal ions for catalysis, and its activity is enhanced by monovalent ions. A minimized, more efficient DNAzyme was also derived. This work demonstrates that highly functional, organic solvent-dependent DNAzymes can be isolated from random-sequence DNA libraries via forced in vitro selection, thus expanding the capability and potential utility of catalytic DNA.

Three on Three: Universal and High-Affinity Molecular Recognition of the Symmetric Homotrimeric Spike Protein of SARS-CoV-2 with a Symmetric Homotrimeric Aptamer.

Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 103 copies/mL. The ELABA was also used to test 50 SARS-CoV-2-positive and 60 SARS-CoV-2-negative patient saliva samples, providing sensitivity and specificity values of 84.0 and 98.3%, respectively, thus highlighting the potential of TMSA52 for the development of future rapid tests.

Cover Feature: Aptamers for SARS-CoV-2: Isolation, Characterization, and Diagnostic and Therapeutic Developments (Anal. Sens. 5/2022).

The cover feature image shows nucleic acid aptamers armed and ready for our battle against the monstrous SARS-CoV-2 virus. Often thought of as "chemical antibodies", these molecular recognition elements are equipped with several unique benefits and have thus been a popular research subject worldwide. Many aptamers for recognizing the spike and nucleocapsid proteins of SARS-CoV-2 have been developed and examined as diagnostic and therapeutic weaponry for the war against COVID-19 and future pandemics. More information can be found in the Review by J. D. Brennan, Y. Li, and co-workers.

Aptamers for SARS-CoV-2: Isolation, Characterization, and Diagnostic and Therapeutic Developments.

The SARS-CoV-2 virus and COVID-19 pandemic continue to demand effective diagnostic and therapeutic solutions. Finding these solutions requires highly functional molecular recognition elements. Nucleic acid aptamers represent a possible solution. Characterized by their high affinity and specificity, aptamers can be rapidly identified from random-sequence nucleic acid libraries. Over the past two years, many labs around the world have rushed to create diverse aptamers that target two important structural proteins of SARS-CoV-2: the spike (S) protein and nucleocapsid (N) protein. These have led to the identification of many aptamers that show real promise for the development of diagnostic tests and therapeutic agents for SARS-CoV-2. Herein we review all these developments, with a special focus on the development of diverse aptasensors for detecting SARS-CoV-2. These include electrochemical and optical sensors, lateral flow devices, and aptamer-linked immobilized sorbent assays.

One Solution for All: Searching for Universal Aptamers for Constantly Mutating Spike Proteins of SARS-CoV-2.

Aptamers that can recognize the spike (S) protein of SARS-CoV-2 with high affinity and specificity are useful molecules towards the development of diagnostics and therapeutics to fight COVID-19. However, this S protein is constantly mutating, producing variants of concern (VoCs) that can significantly weaken the binding by aptamers initially engineered to recognize the S protein of the wildtype virus or a specific VoC. One strategy to overcome this problem is to develop universal aptamers that are insensitive to all or most of the naturally emerging mutations in the protein. We have recently demonstrated this concept by subjecting a pool of S protein-binding DNA aptamers for one-round parallel-SELEX experiments targeting 5 different S protein variants for binding-based sequence enrichment, followed by bioinformatic analysis of the enriched pools. This effort has led to the identification of a universal aptamer that recognizes 8 different variants of the spike protein with equally excellent affinity.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

Invited for the cover of this issue are John Brennan, Yingfu Li, and co-workers at McMaster University. The image depicts MSA52 as a universal DNA aptamer that recognizes spike proteins of diverse SARS-CoV-2 variants of concern. Read the full text of the article at 10.1002/chem.202200078.

A Universal DNA Aptamer that Recognizes Spike Proteins of Diverse SARS-CoV-2 Variants of Concern.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed Kd values ranging from 2 to 10 nM for all variant spike proteins, and also bound similarly to variants not present in the reselection experiments. This aptamer also recognized pseudotyped lentiviruses (PL) expressing eight different spike proteins of SARS-CoV-2 with Kd values between 20 and 50 pM, and was integrated into a simple colorimetric assay for detection of multiple PL variants. This discovery provides evidence that aptamers can be generated with high affinity to multiple variants of a single protein, including emerging variants, making it well-suited for molecular recognition of rapidly evolving targets such as those found in SARS-CoV-2.

Functional Nucleic Acids Under Unusual Conditions.

Functional nucleic acids (FNAs), including naturally occurring ribozymes and riboswitches as well as artificially created DNAzymes and aptamers, have been popular molecular toolboxes for diverse applications. Given the high chemical stability of nucleic acids and their ability to fold into diverse sequence-dependent structures, FNAs are suggested to be highly functional under unusual reaction conditions. This review will examine the progress of research on FNAs under conditions of low pH, high temperature, freezing conditions, and the inclusion of organic solvents and denaturants that are known to disrupt nucleic acid structures. The FNA species to be discussed include ribozymes, riboswitches, G-quadruplex-based peroxidase mimicking DNAzymes, RNA-cleaving DNAzymes, and aptamers. Research within this space has not only revealed the hidden talents of FNAs but has also laid important groundwork for pursuing these intriguing functional macromolecules for unique applications.