Small Efficient Hammerhead Ribozymes
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The hammerhead ribozyme was discovered as a self-cleaving RNA molecule in certain plant viroids and satellite RNAs (
1
). Shortly after its conserved features were defined (
2
,
3
), the hammerhead was shown to be able to act as a true enzyme, cleaving multiple substrates in a bimolecular reaction (
4
). The self-cleaving hammerhead can be divided in a number of ways into two, or even three, separate strands (
4
,
5
). The most useful form has almost all of the conserved nucleotides on the ribozyme strand, leaving minimal sequence requirements in the substrate strand. To be cleavable the substrate must possess the sequence 5′ UH (H is C, U, or A), where cleavage occurs to the 3′ side of H (
6
) (Fig. 1 ). This particular configuration has been the paradigm for hammerhead ribozyme design since 1988. Here we describe variations on this basic design, with the constant theme being to minimize the size of the ribozyme.
Fig. 1
Schematic representation of the hammerhead ribozyme in complex with its complementary substrate. The ribozyme forms helix I with its 5′ arm and the substrate, helix III with its 3′ arm and the substrate, and helix II and loop 2 with the nucleotides joining A
9
and G
12
In the ribozyme, all nucleotides, except for the conserved C
3
to A
9
and G
12
to A
15 1
, may be either ribonucleotides or deoxyribonucleotides The site of cleavage in the substrate is shown by the downward arrow, 3′ to H
17
(H = C, U or A)
