Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities

Table 1 Kinetic parameters for discontinuous ribozymes derived from sTRSV

	[MgCl ₂ ], mM	f _∞	f _a	k _a	k _b	net init rate, min ^-1
RzC [25]	1.0	0.59	0.70	4.23	0.040	1.75
	0.5	0.54	0.58	2.03	0.070	0.65
	0.2	0.42	0.23	3.57	0.091	0.37
	0.1	0.11	0.62	0.92	0.010	0.06
RzC [37]	10	0.74	0.67	6.49	0.013	3.2
	5	0.63	0.77	2.31	0.022	1.1
	2	0.71	0.66	3.52	0.023	1.7
	0.5	0.73	0.38	0.98	0.040	0.3
RzCΔl [37]	10	0.68	0.78	3.92	0.12	2.09
	5	0.76	0.71	5.22	0.033	2.82
	2	0.76	0.57	1.51	0.062	0.67
	0.5	0.58	0.074	1.77	0.028	0.09
RzCΔ2 [37]	10	0.51	0.55	7.1	0.081	2.0
	5	0.66	0.41	3.1	0.025	0.85
	2	0.58	0.229	3.1	0.036	0.43
	0.5	n. d.	n. d.	n. d.	n. d.	n. d.

Kinetic parameters were determined for discontinuous ribozymes as described in Methods. Single-turnover reactions were performed at 25°C or 37°C (square brackets next to ribozyme designations), and the data were fit to a double exponential curve. n.d., no cleavage detected. No cleavage was observed for RzC at 0.075 and 0.05 mM MgCl₂ (not shown).

ISSN: 1471-2091