Molecular Crowding Evolution for Enabling Discovery of Enthalpy-Driven Aptamers for Robust Biomedical Applications

Huang, Mengjiao; Song, Jia; Huang, Peifeng; Chen, Xiaofeng; Wang, Wei; Zhu, Zhi; Song, Yanling; Yang, Chaoyong

doi:10.1021/acs.analchem.9b02697

Cited by 37 publications

(45 citation statements)

References 66 publications

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“…Enthalpy-driven high-affinity aptamers have been developed using molecular crowding evolution. 47 Aptamer screening in a molecular crowding environment (i.e., in blood plasma) both restricts the aptamer's degree of freedom and suppresses the entropic contribution to the binding interaction. Consequently, the binding events that occurred in this environment were mostly enthalpy-driven, so the isolation step predominantly yielded the aptamers that favored noncovalent yet specific binding.…”

Section: Materials Advances Reviewmentioning

confidence: 99%

Detection and beyond: challenges and advances in aptamer-based biosensors

Yoo

2020

Mater. Adv.

169

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Section: Materials Advances Reviewmentioning

confidence: 99%

Detection and beyond: challenges and advances in aptamer-based biosensors

Yoo

2020

Mater. Adv.

169

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“…CTCs from different cancers have been successfully isolated with aptamer-based magnetic separation [ 122 , 123 , 124 ]. For this approach, a magnetic field is applied to separate magnetically tagged target cells from their respective pool [ 125 ].…”

Section: Development Of Cancer-specific Aptamers For Diagnosis and Treatmentmentioning

confidence: 99%

The Potential of Aptamer-Mediated Liquid Biopsy for Early Detection of Cancer

Roy¹,

Pascher

Juratli

et al. 2021

IJMS

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The early detection of cancer favors a greater chance of curative treatment and long-term survival. Exciting new technologies have been developed that can help to catch the disease early. Liquid biopsy is a promising non-invasive tool to detect cancer, even at an early stage, as well as to continuously monitor disease progression and treatment efficacy. Various methods have been implemented to isolate and purify bio-analytes in liquid biopsy specimens. Aptamers are short oligonucleotides consisting of either DNA or RNA that are capable of binding to target molecules with high specificity. Due to their unique properties, they are considered promising recognition ligands for the early detection of cancer by liquid biopsy. A variety of circulating targets have been isolated with high affinity and specificity by facile modification and affinity regulation of the aptamers. In this review, we discuss recent progress in aptamer-mediated liquid biopsy for cancer detection, its associated challenges, and its future potential for clinical applications.

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“…Methods such as surface plasmon resonance (SPR), 36 fluorescent affinity assays, 36 isothermal titration calorimetry (ITC), 34 microscale thermophoresis (MST) 37 are widely utilised. Additionally, to the binding affinity determination, it is observed that the collection of thermodynamics and structural information is required especially for characterising small molecule-aptamer binding.…”

Section: 3-step 2)mentioning

confidence: 99%

Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

Sester¹

2021

Preprint

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Aptamers are synthetic nucleic acid single-stranded oligonucleotides that bind to a wide range of ligands, including cells, proteins, DNA strands, metal ions, and small molecules, with high specificity and affinity. Aptamers have also proven to be highly stable, readily adaptable to chemical modifications, and exhibit reversible binding. As a result, aptamer-based biosensors (aptasensors) are promising replacements for antibody-based biosensors in many applications, particularly for small molecule ligands. This thesis explores an aptamer that binds the drug methamphetamine, and its prospects when incorporated in an electrochemical (e-chem) signal transduction platform. Specifically, we examine the range of interactions between the aptamer and ligand, and with electrodes, and identify a number of challenges in generating robust e-chem aptasensors. Due to their size and limited number of functional groups, further understanding of the aptamer-small molecule ligand interactions is required for the design of future aptasensors – particularly the thermodynamics and structural information about the aptamer-ligand interaction. In fact, detecting small molecules with aptasensors can become challenging because target addition may induce little structural change, and therefore numerous nonspecific interactions may emerge as transduced signals from the biosensor. In this thesis, the combination of spectroscopic and calorimetric analytical techniques reveals a conformational selection binding model, in which binding is entropically driven, and the meth binds via hydrophobic and electrostatic interactions and only induces a modest structural change. This first-of-its-kind study is important for the selection and the design of the aptasensor transduction system. Electrochemical (e-chem) aptasensors offer high inherent sensitivity and practicality as a signal transduction platform. Indeed, different e-chem aptasensor formats have been published before, including labelled and label-free sensors. In screening the viability of three commonly used methods – including labelled and label-free, as well voltammetric and impedance-based methods – we find that each of them suffers from instability of the aptamer-functionalised electrode. This instability compromises our ability to resolve real signals and prompted us to develop ways to understand – and suppress – this baseline drift. The functionalization of the electrode is the critical step in terms of self-assembled monolayer (SAM) stability and SAM aptamer density. Consequently, different protocols of SAMformation were explored and evaluated with respect to stability. We find that instability arises from the uncontrolled arrangement of thiolated aptamers on gold electrodes (including aptamers lying down on the electrode), which is in turn affected by the density of aptamers that can be coupled to the surface. As a consequence, a new protocol is developed using disulfide aptamer pairs to increase the density of correctly tethered aptamers, and generate a stable SAM. Because of the high sensitivity of electrochemical platforms, numerous spurious electrochemical signals may be produced, and controlled for in order to confirm a positive ligand-binding signal. The specificity of the aptamer-target interaction can be checked by testing the response with an interferent molecule, or by substituting the aptamer with a non-binding nucleotide sequence. In this work, these control experiments reveal that target and interferent molecules interact directly (and in different ways) with the bare gold surface, as well as perturbing signals from the aptamer in ways that cannot be linked to a specific aptamer-ligand complex formation. Ultimately, these spurious signals compromise our ability to confirm a real binding signal. The results from this work provide the first clear picture of how an aptamer binds to its small molecule target – which we find is entropically driven, and with only minor structural change induced in the aptamer stem. In addition, the label-free EIS measurements on aptamer SAM electrodes reveal the nature of instabilities, and reveal spurious signals that cannot be sufficiently suppressed at this stage. This knowledge highlights the difficulty in fabricating e-chem aptasensors, and will assist in overcoming challenges faced during research and commercialization of aptasensors area, as well as contributing new insights into troubleshooting, data acquisition, and data validation.

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Molecular Crowding Evolution for Enabling Discovery of Enthalpy-Driven Aptamers for Robust Biomedical Applications

Cited by 37 publications

References 66 publications

Detection and beyond: challenges and advances in aptamer-based biosensors

Detection and beyond: challenges and advances in aptamer-based biosensors

The Potential of Aptamer-Mediated Liquid Biopsy for Early Detection of Cancer

Electrochemical aptasensors: unravelling the critical interactions between aptamers, ligands, and electrodes

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