Key Aspects of Nucleic Acid Library Design for in Vitro Selection

Vorobyeva, Mariya; Davydova, Anna; Vorobjev, P. E.; Пышный, Д. В.; Venyaminova, Alya G.

doi:10.3390/ijms19020470

Cited by 56 publications

(50 citation statements)

References 128 publications

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“…A nucleotide library consisting of 40 bases in the random part was used for screening. This aptamer length is within the recommended range of 30-50 nucleotides, and provides an attractive balance between sequence and fold diversity as well as structural stability [28]. The number of cycles was chosen as a compromise between optimal sequence enrichment and minimal loss of unique sequences, according to the results of previous studies on aptamer dynamics during SELEX [16,27,[29][30][31].…”

Section: Discussionmentioning

confidence: 99%

Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

et al. 2020

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The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8–86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.

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Section: Discussionmentioning

confidence: 99%

Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

et al. 2020

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“…The key aspects of nucleic acid library design were thoroughly reviewed by Vorobyeva et al [17]. All of these factors can alter the selection process to varying extents.…”

Section: Library Designmentioning

confidence: 99%

“…Primer switching during the selection process can also be used to reduce the influence of fixed regions [46]. Most of these protocols have been observed and classified in specific reviews [17,37]. All of these procedures are much more time- and labor-consuming because of the introduction of additional operations, but they have been shown to result in successful aptamer selection.…”

Section: Library Designmentioning

confidence: 99%

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Inside the Black Box: What Makes SELEX Better?

2019

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Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.

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“…Although the basic concept of SELEX is the same, there are several experimental parameters that may affect the overall success for each target [49]. One of the crucial points is the design of the initial nucleic acid library [50].…”

Section: Experimental Design Of Selexmentioning

confidence: 99%

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics

et al. 2019

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Aptamer-based point-of-care (POC) diagnostics platforms may be of substantial benefit in forensic analysis as they provide rapid, sensitive, user-friendly, and selective analysis tools for detection. Aptasensors have not yet been adapted commercially. However, the significance of the applications of aptasensors in the literature exceeded their potential. Herein, in this review, a bottom-up approach is followed to describe the aptasensor development and application procedure, starting from the synthesis of the corresponding aptamer sequence for the selected analyte to creating a smart surface for the sensitive detection of the molecule of interest. Optical and electrochemical biosensing platforms, which are designed with aptamers as recognition molecules, detecting abused drugs are critically reviewed, and existing and possible applications of different designs are discussed. Several potential disciplines in which aptamer-based biosensing technology can be of greatest value, including forensic drug analysis and biological evidence, are then highlighted to encourage researchers to focus on developing aptasensors in these specific areas.

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Key Aspects of Nucleic Acid Library Design for in Vitro Selection

Cited by 56 publications

References 128 publications

Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

Rituximab-specific DNA aptamers are able to selectively recognize heat-treated antibodies

Inside the Black Box: What Makes SELEX Better?

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics

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