Molecular aptamer beacon for myotonic dystrophy kinase-related Cdc42-binding kinase alpha.
A novel strategy for the development of molecular aptamer beacon for a signal transduction protein, myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) was proposed in this work. MRCK is an important downstream effector protein of Cdc42 that phosphorylates proteins involved in organizing actin structures responsible for forming stress fibres, lamellipodia or filopodia. The simple method for MAB design could potentially be applied to other aptamers for modification into a protein probe. The MRCK aptamer was modified into a MAB by adding nucleotides on the 5' end, which are complementary to the 3' end of the aptamer so as to destroy the existing structure and change it into a MB form. In the absence of MRCK, the MAB remained a hairpin structure. However, in the presence of MRCK, the equilibrium was shifted towards the formation of the MRCK-aptamer complex, resulting in the preference for the MRCK-binding conformer, where a fluorescence-quenching pair added to the 5' and 3' ends signaled any protein-dependent conformation change. The development of MABs for signal transduction proteins will have the potential to replace antibodies for diagnostic assays as well as protein studies in cellular imaging.
Tok
Junie
J
Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543, Republic of Singapore.
Lai
Jesyin
J
Leung
Thomas
T
Li
Sam Fong Yau
SF
D052157:Aptamers, Nucleotide / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D005240:Feasibility Studies; D008024:Ligands; D015336:Molecular Probe Techniques; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D012996:Solutions; D013050:Spectrometry, Fluorescence
D052157:Aptamers, Nucleotide; D008024:Ligands; D012996:Solutions; D017346:Protein Serine-Threonine Kinases