5614 Nucleic Acids Research, 2006, Vol. 34, No. 19 existing in vivo (12–15). These interactions, direct evidence for which has been recently derived from the crystallographic structure of a natural hammerhead (16), are currently being incorporated into a new generation of more efﬁcient trans- acting artiﬁcial hammerheads (17–19). Eggplant latent viroid (ELVd) (20) is the last reported member of the family Avsunviroidae of hammerhead viroids (4,21). Both (+) and ( ) ELVd hammerheads display some peculiarities, the inﬂuence of which in the catalytic behavior of these ribozymes has not yet been determined. Here we report the effects of changes in the trinucleotide preceding the self-cleavage site on the self-cleavage kinetics of the ELVd hammerheads, together with a model explaining the lack of trinucleotide AUC in most natural hammerheads, based on differences in co- and post-transcriptional self- cleavage. We also present a comparative analysis of the ELVd hammerheads with other natural hammerheads in a post- transcriptional context at low magnesium concentrations. MATERIALS AND METHODS Construction of hammerhead cDNAs The cDNA of the ELVd (+) hammerhead was PCR-ampliﬁed with Pwo DNA polymerase and the buffer recommended by the supplier (Roche Diagnostics) using as template the plas- mid containing the complete cDNA of the ELVd reference variant (ELVd-2) (20) and the sense RF-689 (50-TAATAC- GACTCACTATAGGCCCCATAGGGTGGTGTG-30) and antisense RF-690 (50-TCTAGACCCCATTTCGACC-30) pri- mers. Mutants of the trinucleotide preceding the self-cleavage site were obtained by PCR ampliﬁcation using the same template and the sense RF-691 (50-TAATACGACTCACT- ATAGGCCCCgTcGGGTGGTGTG-30) and antisense RF-692 (50-TCTAGACCCCgTTTCGACC-30) primers for the hammerhead with the trinucleotide GUC, the sense RF-693 (50-TAATACGACTCACTATAGGCCCCATcGGGTGGTG- TG-30) and antisense RF-690 primers for the hammerhead with the trinucleotide AUC, and the sense RF-694 (50-TA- ATACGACTCACTATAGGCCCCgTAGGGTGGTGTG-30) and antisense RF-692 primers for the hammerhead with the trinucleotide GUA. After an initial denaturation at 94 C for 2 min, the PCR proﬁle (30 cycles) was: 94 C for 20 s, 60 C for 30 s and 72 C for 30 s, with a ﬁnal extension at 72 C for 10 min. The cDNA of the (+) hammerhead of the satellite RNA of tobacco ring spot virus (sTRSV) was prepared by extension of the partially overlapping sense RF-875 (50-GGTAATACGACTCACTATAGGCTGTCACC- GGATGTGCTTTCCGGTC-30) and antisense RF-876 (50-GGGTCTAGACTGTTTCGTCCTCACGGACTCATCAG- ACCGGAAAGC-30) primers (ﬁve cycles at 94 C for 30 s, 30 C for 30 s and 72 C for 30 s, with a ﬁnal extension at 72 C for 2 min). Sense primers included the T7 promoter (underlined) and antisense primers the XbaI site (in italics). Point mutations are indicated with lower case letters. PCR products were separated by PAGE in 10% gels and those with the expected length were eluted, digested with XbaI and cloned into pUC18 opened with XbaI and SmaI. Sequencing of the recombinant plasmids conﬁrmed that they contained only the expected mutations. Construction of (+) hammerheads of peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) has been reported previously (12). Synthesis and purification of hammerhead RNAs Hammerhead RNAs were generated by in vitro transcription of the corresponding recombinant plasmids linearized with XbaI. Transcription reactions (25 ml) contained 40 mM Tris–HCl, (pH 8), 6 mM MgCl2, 2 mM spermidine, 10 mM DTT, 10 mM NaCl, 0.4 mM each of ATP, CTP and GTP, 0.08 mM UTP plus 0.25 mCi/ml [a-32P]UTP, 0.8 U/ml of rRNasin ribonuclease inhibitor (Promega), 20 ng/ml of plas- mid, 1.2 U/ml of T7 RNA polymerase and, when indicated, 0.1–1 mM of the self-cleavage blocking oligodeoxyribo- nucleotide(s). Blocking oligodeoxyribonucleotides were: RF-122 (12) and RF-88 (50-CAAGAGTTTCGTCTCATTTC-30) for PLMVd, RF-142 for CChMVd (12), RF-872 (50-TCTC- ATCAGGGGTGGCACACAC-30) and RF-873 (50-CCCCAT- TTCGACCTTTCG-30) for ELVd, and RF-890 (50-ACTCA- TCAGACCGGAAAGCACATC-30) and RF-891 (50-CTGT- TTCGTCCTCACG-30) for sTRSV. After incubation at 37 C for 45 min, transcription products were mixed with an excess of stop solution [50% formamide, 8 M urea, 50 mM EDTA and 0.25% of bromophenol blue and xylene cyanol], heated at 95 C for 1.5 min, and fractionated by PAGE in 15% gels containing 8 M urea. The uncleaved primary transcripts were extracted from the crushed gel pieces with phenol saturated with buffer [Tris–HCl 10 mM, (pH 8), EDTA 1 mM and SDS 0.7%] containing formamide (40%) to prevent self- cleavage, recovered by ethanol precipitation and resuspended in sterile deionized water. Self-cleavage kinetics of purified hammerhead RNAs To determine the self-cleavage rate constants, the puriﬁed primary transcripts from the different hammerheads were incubated in 20 ml of 50 mM Tris–HCl, (pH 7.5) for 2 min at 95 C followed by a slow ramp down to 25 C at 6 C/min. Samples were incubated for 5 min at 25 C and, after taking a zero-time aliquot, the reactions were initiated by addition of MgCl2 to the indicated concentrations (10 and 50 mM). Aliquots were removed at appropriate time intervals and quenched with 10 volumes of stop solution at 0 C. After heating at 95 C for 1.5 min, primary transcripts and their cleavage products were resolved by PAGE in 15% gels containing 8 M urea and quantiﬁed with a bioimage analyzer. The product fraction at different times (Ft) was determined and ﬁtted to the equation Ft ¼ F1(1 e kt), where F1 is the product fraction at the endpoint, respectively, and k is the ﬁrst order rate constant of self-cleavage (kcat). Values are the means of three indepen- dent experiments. Self-cleavage kinetics of hammerhead RNAs during transcription Reactions, in absence of blocking oligodeoxyribonucleotides, were performed as described above with the exception of the MgCl2 concentration, which was adjusted as indicated in Results, and the T7 RNA polymerase, which was added to trigger the reactions (zero time). Reaction mixtures were pre-incubated at 37 C for 1 min before adding the polymerase and then aliquots were removed at different time intervals