RNase P-Associated External Guide Sequence Effectively Reduces the Expression of Human CC-Chemokine Receptor 5 and Inhibits the Infection of Human Immunodeficiency Virus 1
Figure 2
(a) Cleavage of CCR5 mRNA sequence (substrate ccr5-1) by human RNase P in the presence of different EGSs. No EGS was added to the reaction mixture in lane 1. 1 nM of the EGS C2 (lane 2), C1 (lane 3), and TK1 (lane 4) was incubated with -labeled CCR5 mRNA substrate (20 nM) and human RNase P (2 units) at 37°C in a volume of 10 μL for 15 minutes in buffer A (50 mM Tris, pH 7.4, 100 mM NH4Cl, and 10 mM MgCl2). (b) In vitro binding of -labeled substrate ccr5-1 and EGSs. Substrate ccr5-1 at a concentration of 0.1 nM was incubated either alone (lane 5) or in the presence of 2 nM EGS C1 (lane 7) and C2 (lane 6) in buffer B for 15 min to allow binding and then loaded on a nondenaturing polyacrylamide gel. Experimental details can be found in Section 2.
