A high-throughput assay for a human telomerase protein-human telomerase RNA interaction.
The rapid rate at which cancer cells divide necessitates a mechanism for telomere maintenance, and in approximately 90% of all cancer types the enzyme telomerase is used to maintain the length of telomeric DNA. Telomerase is a multi-subunit enzyme that minimally contains a catalytic protein subunit, hTERT, and an RNA subunit, hTR. Proper assembly of telomerase is critical for its enzymatic activity and therefore is a requirement for the proliferation of most cancer cells. We have developed the first high-throughput screen capable of identifying small molecules that specifically perturb human telomerase assemblage. The screen uses a scintillation proximity assay to identify compounds that prevent a specific and required interaction between hTR and hTERT. Rather than attempting to disrupt all of the individual hTR-hTERT interactions, we focused the screen on the interaction of the CR4-CR5 domain of hTR with hTERT. The screen employs a biotin-labeled derivative of the CR4-CR5 domain of hTR that independently binds [(35)S]hTERT in a functionally relevant manner. The complex between hTERT and biotin-labeled RNA can be captured on streptavidin-coated scintillation proximity beads. Use of 96-well filter plates and a vacuum manifold enables rapid purification of the beads. After optimization, statistical evaluation of the screen generated a Z' factor of 0.6, demonstrating the high precision of the assay.
Keppler
Brian R
BR
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, 27599, USA.
Jarstfer
Michael B
MB
D001710:Biotin / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005163:Factor Analysis, Statistical; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D016337:RNA, Catalytic; D011869:Radioligand Assay / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013462:Sulfur Radioisotopes / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; D013462:Sulfur Radioisotopes; C422952:telomerase RNA; D012313:RNA; D001710:Biotin; D019809:Streptavidin; D019098:Telomerase