Figure 7.

Effect of 3′ Terminal Modifications

(A) Colonies produced by a mixture of 6 × 10⁹ molecules each of the 5′ and 3′ fragments one of which was unmodified while the other was either unmodified at the 3′ terminus (… NPNOH) or modified by periodate oxidation and treatment with aniline (…NP) and subsequent dephosphorylation (…NOH). The mixtures were preincubated for 1 hr at 20°C in a Mg²⁺-free buffer.

(B) Colonies produced by a mixture of 6 × 10⁹ molecules each of the 3′ phosphoryl 5′ fragment and the unmodified 3′ fragment after preincubation for 1 hr at 37°C and indicated Mg²⁺ concentration with or without 0.01 units of CIP. Prior to colony growth, all the samples received a sufficient amount of EDTA to chelate free Mg²⁺ and were then heated for 2 min at 96°C to inactivate phosphatase. RNA colonies were detected by hybridization with the foreign extension-targeted oligonucleotide.

(C) Silver-stained (Igloi 1983) products of 10 min Qβ replicase reactions initiated with 2 ng of a recombinant RNA (type A5, clone 3, cf. Figure 6), which was either unmodified at the 3′ terminus (…NPNOH) or modified by periodate oxidation (…NPNOX) and subsequent treatment with aniline (…NP) and dephosphorylation (…NOH).