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A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

ABSTRACT: Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable in vivo. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity (K _d = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.

SUBMITTER: Le TT

PROVIDER: S-EPMC8159449 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

Le Thao T TT Bruckbauer Andreas A Tahirbegi Bogachan B Magness Alastair J AJ Ying Liming L Ellington Andrew D AD Cass Anthony E G AEG

Chemical science 20200421 17

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable <i>in vivo</i>. We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived ...[more]

PMID: 34122904

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Project description:BACKGROUND:RNA-binding proteins (RBPs) are crucial in modulating RNA metabolism in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Although previous studies on the conservation of RBP targets have been carried out in lower eukaryotes such as yeast, relatively little is known about the extent of conservation of the binding sites of RBPs across mammalian species. RESULTS:In this study, we employ CLIP-seq datasets for 60 human RBPs and demonstrate that most binding sites for a third of these RBPs are conserved in at least 50% of the studied vertebrate species. Across the studied RBPs, binding sites were found to exhibit a median conservation of 58%, ~ 20% higher than random genomic locations, suggesting a significantly higher preservation of RBP-RNA interaction networks across vertebrates. RBP binding sites were highly conserved across primates with weak conservation profiles in birds and fishes. We also note that phylogenetic relationship between members of an RBP family does not explain the extent of conservation of their binding sites across species. Multivariate analysis to uncover features contributing to differences in the extents of conservation of binding sites across RBPs revealed RBP expression level and number of post-transcriptional targets to be the most prominent factors. Examination of the location of binding sites at the gene level confirmed that binding sites occurring on the 3' region of a gene are highly conserved across species with 90% of the RBPs exhibiting a significantly higher conservation of binding sites in 3' regions of a gene than those occurring in the 5'. Gene set enrichment analysis on the extent of conservation of binding sites to identify significantly associated human phenotypes revealed an enrichment for multiple developmental abnormalities. CONCLUSIONS:Our results suggest that binding sites of human RBPs are highly conserved across primates with weak conservation profiles in lower vertebrates and evolutionary relationship between members of an RBP family does not explain the extent of conservation of their binding sites. Expression level and number of targets of an RBP are important factors contributing to the differences in the extent of conservation of binding sites. RBP binding sites on 3' ends of a gene are the most conserved across species. Phenotypic analysis on the extent of conservation of binding sites revealed the importance of lineage-specific developmental events in post-transcriptional regulatory network evolution.

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A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

Publications

A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

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