Sequence Requirements in the Catalytic Core of the “10-23” DNA Enzyme

Abstract

A systematic mutagenesis study of the "10-23" DNA enzyme was performed to analyze the sequence requirements of its catalytic domain. Therefore, each of the 15 core nucleotides was substituted separately by the remaining three naturally occurring nucleotides. Changes at the borders of the catalytic domain led to a dramatic loss of enzymatic activity, whereas several nucleotides in between could be exchanged without severe effects. Thymidine at position 8 had the lowest degree of conservation and its substitution by any of the other three nucleotides caused only a minor loss of activity. In addition to the standard nucleotides (adenosine, guanosine, thymidine, or cytidine) modified nucleotides were used to gain further information about the role of individual functional groups. Again, thymidine at position 8 as well as some other nucleotides could be substituted by inosine without severe effects on the catalytic activity. For two positions, additional experiments with 2-aminopurine and deoxypurine, respectively, were performed to obtain information about the specific role of functional groups. In addition to sequence-function relationships of the DNA enzyme, this study provides information about suitable sites to introduce modified nucleotides for further functional studies or for internal stabilization of the DNA enzyme against endonucleolytic attack.

Cleavage-based signal amplification of RNA

“…Therefore, the first nucleotide 'A' in the catalytic core of the 10-23 DNAzyme from the 5 0 -end was replaced by 'T' to make the cleavage-based RNAamplification strategy feasible. Nevertheless, modification in the catalytic core sequence of the 10-23 DNAzyme usually results in loss of RNA-cleaving catalytic ability [34][35][36] . Fortunately, the modified 10-23 DNAzyme mCat still keep the cleaving ability, even though the mutant site was located in the highly conserved position for the 10-23 catalytic activity 34 ( Fig.…”

Probing Essential Nucleobase Functional Groups in Aptamers and Deoxyribozymes by Nucleotide Analogue Interference Mapping of DNA

“…Consistent with a AÀT base-pair, P and m 6 2 A at position A8, as well as m 3 U and Z at T28 result in diminished ligation activity. Similarly, the interference patterns of C9 and G27 are in agreement with a W.C. base-pair: Z is not tolerated at C9, and m 1 G, m 2 2 G, and P are not accepted at G27.…”

Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

“…This may be because the essential Mg 2+ ion coordinated by the T4–A5 backbone linkage is displaced (Figure S6) 24. We also find position G2, which is highly intolerant to base substitution,19 is largely insensitive to the backbone distorting effects of 2′‐5′ linkages. Thus, our results show that structural mutagenesis through site‐specific insertion of 2′‐5′ linkages can probe novel aspects of nucleic acid structure and function and may be particularly useful for the identification of backbone‐mediated metal‐ion coordination sites.…”

“…These data suggest that T4 and the region C3–G6 in general are sensitive to structural mutagenesis, as well as to base substitutions,19 base deletions,22 or replacement with abasic sites 23. This may be because the essential Mg 2+ ion coordinated by the T4–A5 backbone linkage is displaced (Figure S6) 24.…”

“…Previous analysis had shown that G14 is highly sensitive to mutation as changes to A, C, or T result in around a 20‐fold loss of activity and even conservative substitution of G with inosine or 2‐aminopurine results in a greater than 10‐fold drop in activity 19. In contrast, we find that structural mutagenesis of G14, whereby guanine functional groups are retained but a geometric perturbation of the active site is introduced,20 results in a comparatively mild reduction in 10–23 activity (around fivefold; Figure 4 b,c).…”