Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays.
Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada, L8S 4M1.
Carmen
C
Carrasquilla
Pui Sai
PS
Lau
Yingfu
Y
Li
John D
JD
Brennan
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000302:isolation & purification; D015374:Biosensing Techniques / Q000379:methods; D004355:Drug Stability; D005782:Gels / Q000737:chemistry; D044367:Phase Transition; D012260:Ribonucleases / Q000378:metabolism; D012821:Silanes; D012822:Silicon Dioxide / Q000737:chemistry
D052157:Aptamers, Nucleotide; D005782:Gels; D012821:Silanes; C061205:tetramethoxysilane; D012822:Silicon Dioxide; D012260:Ribonucleases