A nucleic acid switch triggered by the HIV-1 nucleocapsid protein.
A unimolecular oligonucleotide switch, termed here an AlloSwitch, binds the mature HIV-1 nucleocapsid protein, NCp7. This switch can be used as an indicator for the presence of free NCp7 and NC domains in precursor and fusion proteins. It is thermodynamically stable in two conformations, H and O. A FRET pair is covalently attached to the strands to report on the molecular state of the switch. The results show that NC has an affinity for O 170 times higher than its affinity for H and that in the absence of NC the equilibrium ratio K1 = [O]/[H] = 0.10 +/- 0.03 for the switch sequence reported here. The change between the two states happens on a rapid kinetic time scale. A framework is introduced to aid in the design of AlloSwitches aimed at other targets. A high-affinity probe segment must be available to bind the target in the O-form, while a cover segment hides the probe in H. A key is adjusting the cover sequence to favor the H-form by a factor of 10-1000. This affords a robust response to small changes in target concentration, while saturation produces more than 90% of the maximal change in fluorescence. When a competitor displaces the switch from the NC-O complex, the released switch reverts to the H-form. This is the basis for a mix-and-read strategy for high-throughput screening of anti-nucleocapsid drug candidates that is much simpler to execute than traditional assays that require immobilization and washing steps.
Department of Chemistry, Syracuse University, Syracuse, New York 13244, USA.
Christopher L
CL
DeCiantis
Danielle K
DK
Jensen
Bruce S
BS
Hudson
Philip N
PN
Borer
D036022:Capsid Proteins; D031541:Fluorescence Resonance Energy Transfer; D015683:Gene Products, gag; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D050506:Mutant Chimeric Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; D008024:Ligands; D050506:Mutant Chimeric Proteins; C071884:NCP7 protein, Human immunodeficiency virus 1; D019590:Nucleocapsid Proteins; D009841:Oligonucleotides; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D014364:Tryptophan