DNAmoreDB - A Database of Deoxyribozymes

Published on 1997 in Chem. Biol. volume 4 issue 8.

PubMed ID: 9281526

DOI:10.1016/s1074-5521(97)90244-1

Abstract:

BACKGROUND:
RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors.

RESULTS:
We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a 'first generation' of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates approximately 10(7)-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded 'second generation' DNAzymes that self-cleaved at rates of approximately 0.01 min-1 (a 10(8)-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes.

CONCLUSIONS:
Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.



DNAzymes linked to this article:

Name Isolated sequence Length Reaction
G1 TTTACCCAAGAAGGGGTGCGTACTTTACTTGCGAT      35 RNA cleavage
G2 TTTACCCAAGAAGGGGTGCGTACTTACTTGC      31 RNA cleavage
G3 TTTACCCAAGAAGGGGTGCGTACTATGCTACCTTATTAACG      41 RNA cleavage
G4 TTTACCCAAGAAGGGGTGCGTACTATGCTACCTTATGAC      39 RNA cleavage
G5 TTTACCCAAGAAGGGGTGCGTACTATGCTACCTTATGACGT      41 RNA cleavage
G6 TTTACCCAAGAAGGGGTGCGTACTATACTACTC      33 RNA cleavage
G7 TTTACCCAAGAAGGGGTGGCATGAGCGCTACCCAGCACA      39 RNA cleavage
G8 TTTACCCAAGAAGGGGTGGCTAGAGCGCTACCCAGCAGA      39 RNA cleavage
C1 CCCCAACTTGAATCCGGTTCGCAAAGTTGCTTACATCTC      39 RNA cleavage
T1 CCTGCGCACTCCGGTTCGTTCCGGCACTATTTATTCAAG      39 RNA cleavage
T2 CCTGCGCACTCCGGTTCGTTCCGGCACTATTTATTCATC      39 RNA cleavage
T3 CCTGCGCACTCCGGTTCGTTCCGGCACTATTTATTCAT      38 RNA cleavage
T4 CCTGCTTCTCCGGTTCGTTACCGACATCATTCATT      35 RNA cleavage
Na2 TTTACCCAAGAAGGGGTGCCTACTtTGCCACCATATT      37 RNA cleavage
Na3 TTTACCCAAGAAGGGGTGCGTTCAATGCTACCTTATTAAC      40 RNA cleavage
Na4 TTTACCCAAGAAGGGGTGGACTGAGCGCCTACCCAGCAGAG      41 RNA cleavage
Na6 TTTACCCAAGAAGGGGTGCTACTATGTTACCTTATTACG      39 RNA cleavage
Na9 TTTACCCAAGAAGGGGTGCTTCACTATGCTACGTAATTAAC      41 RNA cleavage
Na10 TTTACCCAAGAAGGGGTGCGTACTATAGCGACCGTATTAAC      41 RNA cleavage
Na13 TTTACCCAAGAAGGGGTGCGCACTAGACTACCTTATAACT      40 RNA cleavage
Na15 TTTACCCAAGAAGGGGTGCGGACTTGGCTTCATTATTAATTGA      43 RNA cleavage
Na19 TTTACCCAAGAAGGGGTGCTACTAAGCCTACTTTATTACC      40 RNA cleavage
Na14 TTTACCCAAGAAGGGGTGCTACATGTACTTATAACTTCGA      40 RNA cleavage
Na16 CCCAAGAAGGGGTGCGTAAACTGCTACCCTATTAACCTA      39 RNA cleavage
Na1 TTTGTACCCAAGAAGGGGTGATACTAGGCTACCGTGTTAACG      42 RNA cleavage
Na18 TTTGACCCAAGAAGGGGTGCATACTTAGCTGACCTTATTCAAC      43 RNA cleavage
Na22 TTTACCCAAGAAGGGGTGCCTACAATGCTACCTGTTCACG      40 RNA cleavage
Na23 TTTACCCAATAAGGGGTGCCTACTCTGCTACGTTATTTAACG      42 RNA cleavage
Na17 TTACCCAAGAAGGGGTGCCTAATTGCTACTCACAAAC      37 RNA cleavage
Na8 TTGACCCAAGAAGGGGTGACTACTTTGCTACGTTATTCCAC      41 RNA cleavage
Na24 TTTACCCAAGAAGGGGTGCAACCATTGCCTAACCTAATCACCG      43 RNA cleavage
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