Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer.
Biochem Biophys Res Commun
; 711: 149908, 2024 Jun 04.
Article
in En
| MEDLINE
| ID: mdl-38613867
ABSTRACT
RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Aptamers, Nucleotide
Language:
En
Journal:
Biochem Biophys Res Commun
Year:
2024
Document type:
Article
Affiliation country:
Canada
Country of publication:
United States