are very much inﬂuenced by tertiary interactions between loops 1 and 2 (12–15), we designed two oligodeoxyribo- nucleotides, one complementary to the complete loop 1 and its adjacent sequences of helices I and II, and the other com- plementary to the complete loop 2 and its adjacent sequences of helices II and III (Figure 2A). Co-incorporation of the two oligodeoxyribonucleotides to the in vitro transcription mix- ture abolished essentially self-cleavage (Figure 2B, compare lanes 1 and 3 with 2), providing further support for the pro- posed tertiary interactions between loops 1 and 2. Moreover, the same approach also worked for the PLMVd (+) and sTRSV (+) hammerheads (data not shown), thus indicating that it may be of general application. A possible explanation for these results is that loops 1 and 2 may be more easily accessible to antisense oligodeoxyribonucleotide than regions more structured like the central conserved core or its ﬂanking helices. Self-cleavage kinetics of the ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site The trinucleotide preceding the self-cleavage site of most natural hammerheads, including the ELVd ( ) hammerhead, is GUC (3,20). However, there are exceptions to this rule. As already indicated, the trinucleotide preceding the self- cleavage site of the ELVd (+), sCYDV-RPV (+) and csc RNAs ( ) hammerheads is AUA (20,27–29). Furthermore, the trinucleotide preceding the self-cleavage site of the (+) hammerhead of csc RNAs and the ( ) hammerhead of the Nucleic Acids Research, 2006, Vol. 34, No. 19 5617 satellite RNA of lucerne transient streak virus (sLTSV) is GUA (5,28,29) (Table 1). Because the effects on self-cleavage kinetics of interchanging these trinucleotides preceding the self-cleavage site have not been determined in a cis context and at low magnesium concentration, we examined this ques- tion in the ELVd (+) hammerhead. As a control, we also stud- ied the effect of the trinucleotide AUC, which with one exception (see below), has never been found preceding the self-cleavage site of natural hammerheads. At very low magnesium concentration (10 mM), the ELVd- (+)-GUC mutant (where the GUC refers to the trinucleotide preceding the self-cleavage site) presented a self-cleavage rate constant signiﬁcantly higher than the wild-type ELVd- (+)-AUA hammerhead (Figure 3A). The ELVd-(+)-GUA mutant had a self-cleavage rate constant intermediate between those of the ELVd-(+)-GUC mutant and the ELVd- (+)-AUA wild-type but, surprisingly, the ELVd-(+)-AUC mutant displayed the highest rate constant (Figure 3A). More- over, at the end-point of the reaction, 95% of molecules from the three mutants with the highest rate constants self- cleaved, whereas this fraction was only 60% for the wild-type ELVd-(+)-AUA hammerhead (Figure 3A). A similar trend in the rate constants was observed when the magnesium concen- tration was increased moderately (50 mM): ELVd-(+)- AUC ELVd-(+)-GUC>ELVd-(+)-GUA>ELVd-(+)-AUA, with the fraction of self-cleaved molecules at the end-point of the reaction being higher than 95% in all cases (Figure 3B). At higher magnesium concentration (500 mM), the self-cleavage rates were too fast to be analyzed manually (self-cleavage was essentially complete after 30 s, data not Figure 3. Representative self-cleavage kinetics at 10 mM (A) and 50 mM (B) magnesium of the purified wild-type ELVd (+) hammerhead and of three mutants affecting the trinucleotide preceding the self-cleavage site. The fraction of product at different times (Ft) was determined by radioactivity quantitation of the corresponding gel bands with a bioimage analyzer and fitted to the equation Ft ¼ F1 (1 e kt), where F1 is the fraction of product at the endpoint of the reaction and k the first order rate constant of cleavage (kcat). The inset displays the first 2 min for each experiment. The mean values obtained for the rate constants of the four hammerheads and for F1 (between parenthesis) are shown below. The concentration of Mg was 1000-fold higher than that of hammerheads.