Simple Methods and Rational Design for Enhancing Aptamer Sensitivity and Specificity

Abstract

Aptamers are structured nucleic acid molecules that can bind to their targets with high affinity and specificity. However, conventional SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods may not necessarily produce aptamers of desired affinity and specificity. Thus, to address these questions, this perspective is intended to suggest some approaches and tips along with novel selection methods to enhance evolution of aptamers. This perspective covers latest novel innovations as well as a broad range of well-established approaches to improve the individual binding parameters (aptamer affinity, avidity, specificity and/or selectivity) of aptamers during and/or post-SELEX. The advantages and limitations of individual aptamer selection methods and post-SELEX optimizations, along with rational approaches to overcome these limitations are elucidated in each case. Further the impact of chosen selection milieus, linker-systems, aptamer cocktails and detection modules utilized in conjunction with target-specific aptamers, on the overall assay performance are discussed in detail, each with its own advantages and limitations. The simple variations suggested are easily available for facile implementation during and/or post-SELEX to develop ultrasensitive and specific assays. Finally, success studies of established aptamer-based assays are discussed, highlighting how they utilized some of the suggested methodologies to develop commercially successful point-of-care diagnostic assays.

G-Quadruplex-Forming DNA Aptamers Inhibit the DNA-Binding Function of HupB and Mycobacterium tuberculosis Entry into Host Cells

“…The polyanionic compound DexSO 4 was used to mask non-specific charge-based interactions of ssDNA aptamers, which are naturally polyanionic in nature, 21 , 22 with non-target molecules having a positive charge. To assess the specificity of the four selected HupB aptamers, ALISA was carried out with a panel of Mtb antigens in the presence and absence of 500 nM DexSO 4 and compared to that of previously in-house generated polyclonal and monoclonal anti-HupB antibodies by ELISA.…”

The Role of CYP450 Drug Metabolism in Precision Cardio-Oncology

“…This leads to cleavage of the mRNA-DNA heteroduplex by RNase H endonuclease, which prevents translation of the target mRNA and thereby downregulates expression of the corresponding target protein [103]. Aptamers are single-stranded oligonucleotides that bind target mRNA with high affinity and specificity, through physiochemical mechanisms such as hydrophobic, electrostatic, hydrogen bonding, van der Waals forces, base stacking and shape complementarity interactions [104,105]. Due to their desirable tissue penetrability and high affinity and specificity target-binding, aptamers are expected to become a widely used platform for delivery of therapeutic small RNAs [105].…”

The Use of Aptamers and Molecularly Imprinted Polymers in Biosensors for Environmental Monitoring: A Tale of Two Receptors

“…When compared to aptamers, MIPs can be rationally designed in a bottom-up mannor using a number of techniques, including computational modelling and experimental methods [20,21]. Aptamers, on the other hand, can only undergo rational design after their selection (post-selection modification) [22].…”