RNA Aptamers for a tRNA-Binding Protein from Aeropyrum pernix with Homologous Counterparts Distributed Throughout Evolution

In the present in vitro selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon Aeropyrum pernix. Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for A. pernix Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3′-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3′ terminus of tRNA in the interaction with Trbp-like structures.

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2′-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP)

International Journal of Molecular Sciences ◽

10.3390/ijms19123883 ◽

2018 ◽

Vol 19 (12) ◽

pp. 3883 ◽

Cited By ~ 5

Author(s):

Jasmin Aldag ◽

Tina Persson ◽

Roland Hartmann

Keyword(s):

In Vitro Selection ◽

Binding Protein ◽

High Specificity ◽

Rna Aptamers ◽

Pyrimidine Nucleotides ◽

Consensus Sequences ◽

Structure Probing ◽

Potential Applications ◽

Nuclease Degradation

Lipopolysaccaride binding protein (LBP), a glycosylated acute phase protein, plays an important role in the pathophysiology of sepsis. LBP binds with high affinity to the lipid part of bacterial lipopolysaccaride (LPS). Inhibition of the LPS-LBP interaction or blockage of LBP-mediated transfer of LPS monomers to CD14 may be therapeutical strategies to prevent septic shock. LBP is also of interest as a biomarker to identify septic patients at high risk for death, as LBP levels are elevated during early stages of severe sepsis. As a first step toward such potential applications, we isolated aptamers specific for murine LBP (mLBP) by in vitro selection from a library containing a 60-nucleotide randomized region. Modified RNA pools were transcribed in the presence of 2′-fluoro-modified pyrimidine nucleotides to stabilize transcripts against nuclease degradation. As verified for one aptamer experimentally, the selected aptamers adopt a “three-helix junction” architecture, presenting single-stranded 7-nt (5′-YGCTTCY) or 6-nt (5′-RTTTCY) consensus sequences in their core. The best binder (aptamer A011; Kd of 270 nM for binding to mLBP), characterized in more detail by structure probing and boundary analysis, was demonstrated to bind with high specificity to murine LBP.

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In vitro selection of RNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus: A possible role of GC-rich RNA motifs in NS5B binding

Virology ◽

10.1016/j.virol.2009.02.032 ◽

2009 ◽

Vol 388 (1) ◽

pp. 91-102 ◽

Cited By ~ 5

Author(s):

Hiroshi Kanamori ◽

Kazuhito Yuhashi ◽

Yasutoshi Uchiyama ◽

Tatsuhiko Kodama ◽

Shin Ohnishi

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

In Vitro Selection ◽

Rna Aptamers ◽

Rna Dependent Rna Polymerase ◽

Rna Motifs ◽

Selection Of

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In vitro selection and characterization of RNA aptamers binding thyroxine hormone

Biochemical Journal ◽

10.1042/bj20061216 ◽

2007 ◽

Vol 403 (1) ◽

pp. 129-138 ◽

Cited By ~ 45

Author(s):

Dominique Lévesque ◽

Jean-Denis Beaudoin ◽

Sébastien Roy ◽

Jean-Pierre Perreault

Keyword(s):

In Vitro Selection ◽

Biochemical Characterization ◽

Rna Aptamers ◽

Rna Structures ◽

Chemical Probing ◽

Guanine Quadruplex ◽

Exponential Enrichment ◽

Helical Region

RNA possesses the ability to bind a wide repertoire of small molecules. Some of these binding interactions have been shown to be of primary importance in molecular biology. For example, several classes of mRNA domains, collectively referred to as riboswitches, have been shown to serve as RNA genetic control elements that sense the concentrations of specific metabolites (i.e. acting as direct sensors of chemical compounds). However, to date no RNA species binding a hormone has been reported. Here, we report that the use of an appropriate SELEX (systematic evolution of ligands by exponential enrichment) strategy results in the isolation of thyroxine-specific aptamers. Further biochemical characterization of these aptamers, including mutational studies, the use of transcripts with site-specific modified nucleotides, nuclease and chemical probing, binding-shift assays and CD, demonstrated that these RNA structures included a G-rich motif, reminiscent of a guanine quadruplex structure, adjacent to a helical region. The presence of the thyroxine appeared to be essential for the formation of the structural motif's scaffold. Moreover, the binding is shown to be specific to thyroxine (T4) and tri-iodothyronine (T3), the active forms of the hormone, whereas other inactive derivatives, including thyronine (T0), do not support complex formation. These results suggest that this aptamer specifically binds to the iodine moieties of the thyroxine, a previously unreported ability for an RNA molecule.

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