High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA

Abstract

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.

High affinity ligands from in vitro selection: Complex targets

“…The proteins and nucleic acids were precipitated in 0.3 M NaOA c and 2 vol of ethanol, pelleted by centrifugation, and analyzed by SDS͞ PAGE (4-12% polyacrylamide) with staining of the proteins by silver. ssDNA because we had not yet decided what modified RNA library was likely to be most useful for later experiments (32)(33)(34). A pool of 10 13 ssDNA molecules containing a 30-base random region flanked by fixed sequences required for the hybridization of primers was incubated with the RBC ghosts.…”

Selection of a novel DNA thioaptamer against HER2 structure

“…However, unmodified oligonucleotides are susceptible to hydrolysis by nucleases, limiting their potential for in vivo biomedical applications. To improve aptamers' function as tumor-targeting ligands, various chemical modifications for enhancing their stability have been introduced [28][29][30][31][32]. Among these modification methods, phosphate backbone modification with phosphorothioate is a promising strategy, since it does not hamper the target-binding affinity of the oligonucleotides.…”

Smart functional nucleic acid chimeras: Enabling tissue specific RNA targeting therapy

“…126 Stemming from their initial enzymatic recognition studies, 2 0 -amino pyrimidines, 2 0 -fluoro pyrimidines and 2 0 -O-Methyl nucleotides have been successfully applied in aptamer development by conventional SELEX-based methodologies. [127][128][129][130][131][132][133][134] LNA is one of the successful nucleotide analogs extensively utilized in various fields because of their remarkable properties. 113,114 In LNA the sugar ring is conformationally locked by a O2 0 -C4 0 methylene linkage to adopt N-type sugar puckering.…”