Hammerhead ELVd (+) ELVd ( ) ELVd-(+)-GUC ELVd-(+)-AUC ELVd-(+)-GUA PLMVd (+) CChMVd (+) sTRSV (+) cscRNA (+) cscRNA ( ) sLTSV ( ) sCYDV-RPV (+) 7/4 7/4 7/4 7/4 7/4 5/3 6/7 6/7 6/6 6/6 6/13 4/8 4/4 4/4 4/4 4/4 4/4 5/6 5/6 4/4 4/4 4/4 4/6 Atypicalb 6 5 6 6 6 5 7 4 7 5 5 6 5616 Nucleic Acids Research, 2006, Vol. 34, No. 19 Table 1. Structural characteristcs of some hammerhead ribozymes Helix I/ loop 1a Helix II/ loop 2 Helix III Trinucleotide preceding the self-cleavage site (50!30) AUA GUC GUC AUC GUA GUC GUC GUC GUA AUA GUA AUA aLength of helices (in base pairs) and loops (in bases). bLong helix II composed by segments of 5, 4 and 4 bp separated by internal loops and closed by a terminal loop of 4 bases. The sequence heterogeneity observed in natural ELVd variants is consistent with the existence in helix I of both ELVd hammerheads of a base pair between positions 1.7 and 2.7, because a U–G pair is substituted by a U–A pair or viceversa (20). In the (+) ELVd hammerhead, positions 15.2 and 16.2 form a U–A pair instead of the predominant C–G pair, a situation also found in the (+) hammerhead of satellite RNA of cereal yellow dwarf virus RPV (sCYDV- RPV) (27) and in the ( ) hammerhead of two cherry small circular RNAs (csc RNAs) (28,29). In these three hammer- heads the residue preceding the self-cleavage site is A and not C (Table 1) as in most natural hammerheads, suggesting some relationship between the three positions (20,28,29). The two ELVd hammerheads are more related to each other than to other hammerheads, as also occurs between the hammer- heads of other viroid and viroid-like RNAs (5,25,26,28,29). Antisense oligodeoxyribonucleotides against loops 1 and 2 efficiently prevent self-cleavage during in vitro transcription of minimal natural hammerheads Although the self-cleavage rates of artiﬁcial hammerheads can be estimated based on measurements from in vitro tran- scription reactions (12,22,30,31), most natural hammerheads self-cleave efﬁciently under standard in vitro transcriptions. This forces the adoption of non-standard conditions, particu- larly low pH and magnesium concentration (12,31), which complicate further kinetic comparisons between different hammerheads. The alternative approach for estimating ham- merhead self-cleavage rates, isolation of the uncleaved RNA from in vitro transcriptions and its subsequent incubation in a protein-free buffer (1,32–34), suffers the limitation that the uncleaved primary transcript usually represents a minor fraction of the transcription products. Extensive self-cleavage of the hammerhead RNA during in vitro transcription can be avoided by incorporating an oligodeoxyribonucleotide com- plementary to part of the ribozyme. This approach has been previously used with one ribozyme of hepatitis delta virus RNA (35), with in vitro selection of self-cleaving RNAs (36), with the CChMVd (+) and PLMVd (+) hammerheads (12) Figure 2. (A) Positions of the ELVd (+) hammerhead covered by two antisense oligodeoxyribonucleotides (in color). Other details as in the legend to Figure 1. (B) Self-cleavage during transcription of the ELVd (+) hammerhead in the presence and absence of the antisense oligodeoxyribo- nucleotides (lanes 1 and 2, respectively), and of the mutant ELVd-(+)-GUC hammerhead (in which the wild-type AUA trinucleotide preceding the self- cleavage site has been substituted by GUC) in the presence of the antisense oligodeoxyribonucleotides (lane 3). Lane 4, RNA markers of 65 and 55 nt. Aliquots of the reaction mixtures were analyzed by PAGE in 15% gels containing 8 M urea that were stained with ethidium bromide. Positions of the complete (C) primary transcript (60 nt) and of the resulting self-cleavage 30 fragment (30F) (51 nt) are indicated with arrows. The self-cleavage 50 fragment (9 nt) migrated out of the gel. and with certain hammerheads encoded in the genome of Arabidopsis thaliana (37). In the CChMVd case, high levels of uncleaved primary transcripts (75% of the total RNA) were obtained in the presence of oligodeoxyribonucleotides complementary to the sequences forming part of helix I/ loop 1 and helix II/loop 2 and the connecting CUGAUGA of the conserved central core (12). Preliminary experiments with the ELVd (+) hammerhead showed that self-cleavage proceeded extensively during in vitro transcription under standard conditions (data not shown). In an attempt to attenuate this problem, an antisense oligodeoxyribonucleotide analogous to those used previously with the CChMVd (+) hammerhead (12) was incorporated to the in vitro transcription mixture. However, the protecting effects were limited, as also were the effects of two other oligodeoxyribonucleotides complementary to sequences form- ing part of helix I/loop 1 and helix III connected by the A pre- ceding the self-cleavage site, and to sequences forming part of helix II/loop 2 and helix III (data not shown). Considering that the fast self-cleavage rates of most natural hammerheads