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OALib Journal期刊
ISSN: 2333-9721
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Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

DOI: 10.1186/1472-6769-4-1

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Abstract:

A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent.Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.Hammerhead ribozymes were discovered as self-cleaving motifs in a number of small, circular, pathogenic RNAs in plants [1-3]. Uhlenbeck [4] showed that the ribozyme was able to act in a bimolecular fashion as a true enzyme, ie each ribozyme was able to cleave multiple substrates. Haseloff and Gerlach [5] divided the hammerhead into a form in which the majority of the conserved nucleotides were located on the enzyme strand, with the only sequence requirements for the substrate being UH (H = A, U or C) [6-8]. Since 1988 this configuration, as shown in Figure 1, has been the paradigm for hammerhead ribozyme design. Hammerhead ribozymes are sequence-specific RNA cleaving agents with the potential to control the expre

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