Optimization Of Aptamers Research Articles

RaptGen-Assisted Generation of an RNA/DNA Hybrid Aptamer against SARS-CoV-2 Spike Protein.

Optimization of aptamers in length and chemistry is crucial for industrial applications. Here, we developed aptamers against the SARS-CoV-2 spike protein and achieved optimization with a deep-learning-based algorithm, RaptGen. We conducted a primer-less SELEX against the receptor binding domain (RBD) of the spike with an RNA/DNA hybrid library, and the resulting sequences were subjected to RaptGen analysis. Based on the sequence profiling by RaptGen, a short truncation aptamer of 26 nucleotides was obtained and further optimized by a chemical modification of relevant nucleotides. The resulting aptamer is bound to RBD not only of SARS-CoV-2 wildtype but also of its variants, SARS-CoV-1, and Middle East respiratory syndrome coronavirus (MERS-CoV). We concluded that the RaptGen-assisted discovery is efficient for developing optimized aptamers.

Optimization of Gonyautoxin1/4-Binding G-Quadruplex Aptamers by Label-Free Surface-Enhanced Raman Spectroscopy

Nucleic acids with G-quadruplex (G4) structures play an important role in physiological function, analysis and detection, clinical diagnosis and treatment, and new drug research and development. Aptamers obtained using systematic evolution of ligands via exponential enrichment (SELEX) screening technology do not always have the best affinity or binding specificity to ligands. Therefore, the establishment of a structure-oriented experimental method is of great significance. To study the potential of surface-enhanced Raman spectroscopy (SERS) in aptamer optimization, marine biotoxin gonyautoxin (GTX)1/4 and its G4 aptamer obtained using SELEX were selected. The binding site and the induced fit of the aptamer to GTX1/4 were confirmed using SERS combined with two-dimensional correlation spectroscopy. The intensity of interaction between GTX1/4 and G4 was also quantified by measuring the relative intensity of SERS bands corresponding to intramolecular hydrogen bonds. Furthermore, the interaction between GTX1/4 and optimized aptamers was analyzed. The order of intensity change in the characteristic bands of G4 aptamers was consistent with the order of affinity calculated using microscale thermophoresis and molecular dynamics simulations. SERS provides a rapid, sensitive, and economical post-SELEX optimization of aptamers. It is also a reference for future research on other nucleic acid sequences containing G4 structures.

In silico predictions and optimization of aptamers against Streptococcus agalactiae surface protein using computational docking

Abstract Aptamers are single-stranded RNA or DNA oligonucleotides that form unique three-dimensional (3D) structures that can bind a wide range of molecules and compounds The development of aptamers against targeted biomolecules using computational approach is a novel method to obtain viable chemical-based antibody substitutes. The conventional method to derive aptamers using SELEX technology involves multiple rounds of selection and enrichment, which is laborious, costly and time-consuming. It is also inefficient as it will often fail to isolate high binding aptamers. We evaluated a new approach to improve aptamer designs by using in silico refinement of the desired binding requirement on the biomolecule target. The three-dimensional (3D) structures of single strand oligonucleotides were modelled to contain hairpin(s) and predicted to retain folding stability base on free energy secondary structure formation. The size and number of hairpins were then optimized with aptamer sequence length to derive the highest binding score. We utilized the method against Streptoccocus agalactiae surface protein as test model and generated binding score values by molecular docking using AutoDock Vina. The aptamer binding affinities were then evaluated for binding capacity improvement via specific structural design. Molecular docking experiments indicated the feasibility of using in silico technique to select aptamers that can function as synthetic antibodies and revealed 3D structural conformity as an essential requirement in aptamer design.

Nucleic acids delivering nucleic acids.

Nucleic acid therapeutics, including siRNAs, miRNAs/antimiRs, gRNAs and ASO, represent innovative and highly promising molecules for the safe treatment of a wide range of pathologies. The efficiency of systemic treatments is impeded by 1) the need to overcome physical and functional barriers in the organism, and 2) to accumulate in the intracellular active site at therapeutic concentrations. Although oligonucleotides either as modified naked molecules or complexed with delivery carriers have revealed to be effectively delivered to the affected target cells, this is restricted to topic treatments or to a few highly vascularized tissues. Therefore, the development of effective strategies for therapeutic nucleic acid selective delivery to target tissues is of primary importance in order to reduce the occurrence of undesired effects on non-target healthy tissues and to permit their translation to clinic. Due to their high affinity for specific ligands, high tissue penetration and chemical flexibility, short single-stranded nucleic acid aptamers are emerging as very attractive carriers for various therapeutic oligonucleotides. Yet, different aptamer-based bioconjugates, able to provide accumulation into target tissues, as well as efficient processing of therapeutic oligonucleotides, have been developed. In this respect, nucleic acid aptamer-mediated delivery strategies represent a powerful approach able to increase the therapeutic efficacy also highly reducing the overall toxicity. In this review, we will summarize recent progress in the field and discuss achieved objectives and optimization of aptamers as delivery carriers of short oligonucleotides.

Multiple Different Strategies for Post-SELEX Optimization of Aptamers

Multiple Different Strategies for Post-SELEX Optimization of Aptamers

Aptamer evolution for array-based diagnostics

Closed loop aptameric directed evolution, (CLADE) is a technique enabling simultaneous discovery, evolution, and optimization of aptamers. It was previously demonstrated using a fluorescent protein, and here we extend its applicability with the generation of surface-bound aptamers for targets containing no natural fluorescence. Starting from a random population, in four generations CLADE produced a new aptamer to thrombin with high specificity and affinity. The best aptameric sequence was void of the set of four guanine repeats typifying thrombin aptamers and, thus, highlights the benefits of evolution performed in an environment closely mimicking the final diagnostic application.