Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine.

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Zinc fingers as protein recognition motifs: Structural basis for the GATA-1/Friend of GATA interaction

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0407511102 ◽

2005 ◽

Vol 102 (3) ◽

pp. 583-588 ◽

Cited By ~ 68

Author(s):

C. K. Liew ◽

R. J. Y. Simpson ◽

A. H. Y. Kwan ◽

L. A. Crofts ◽

F. E. Loughlin ◽

...

Keyword(s):

Zinc Fingers ◽

Structural Basis ◽

Protein Recognition ◽

Recognition Motifs

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Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1309538110 ◽

2013 ◽

Vol 110 (40) ◽

pp. 15967-15972 ◽

Cited By ~ 77

Author(s):

Y.-W. Cheung ◽

J. Kwok ◽

A. W. L. Law ◽

R. M. Watt ◽

M. Kotaka ◽

...

Keyword(s):

Lactate Dehydrogenase ◽

Dna Aptamer ◽

Structural Basis

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Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

Essays in Biochemistry ◽

10.1042/ebc20150004 ◽

2016 ◽

Vol 60 (1) ◽

pp. 27-35 ◽

Cited By ~ 15

Author(s):

Pawan Jolly ◽

Pedro Estrela ◽

Michael Ladomery

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Rna Aptamers ◽

Locked Nucleic Acid ◽

Synthetic Analogue ◽

Dna And Rna ◽

Naturally Occurring ◽

Wide Range ◽

Synthetic Oligonucleotides ◽

Shed Light

There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications.

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Selection of DNA aptamers with two modified bases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615475114 ◽

2017 ◽

Vol 114 (11) ◽

pp. 2898-2903 ◽

Cited By ~ 89

Author(s):

Bharat N. Gawande ◽

John C. Rohloff ◽

Jeffrey D. Carter ◽

Ira von Carlowitz ◽

Chi Zhang ◽

...

Keyword(s):

Chemical Diversity ◽

Rna Aptamers ◽

Dna Aptamers ◽

Dramatic Improvement ◽

Protein Targets ◽

Dna And Rna ◽

Modified Dna ◽

Pyrimidine Bases ◽

Representative Protein ◽

Selection Of

The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid–like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.

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Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization

10.1101/268458 ◽

2018 ◽

Author(s):

Ahmed M. Malik ◽

Roberto A. Miguez ◽

Xingli Li ◽

Ye-Shih Ho ◽

Eva L. Feldman ◽

...

Keyword(s):

Nucleic Acid ◽

Self Assembly ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nucleic Acid Binding ◽

Rna Recognition Motifs ◽

Matrin 3 ◽

Dna And Rna ◽

Acid Processing ◽

Recognition Motifs

ABSTRACTAbnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3’s zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on neuronal survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.

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