Novel approach to analyzing RNA aptamer-protein interactions: toward further applications of aptamers.
Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. DeltaNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the deltaNS3 protein in this study (KD = 4 x 10(-8)). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.
Hwang
Joonsung
J
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Nishikawa
Satoshi
S
D052157:Aptamers, Nucleotide; D015374:Biosensing Techniques; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D052157:Aptamers, Nucleotide; D012313:RNA; D020365:RNA Helicases; D012367:RNA, Viral; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C503099:G925-s50 RNA aptamer; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases