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Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme.

Schlosser K, Gu J, Sule L, Li Y - Nucleic Acids Res. (2008)

Bottom Line: In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8-17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions.We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5' rN(18)-N(1.1) 3') cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3' position (N(1.1)) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5' position (rN(18)) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity.Three optimal 8-17 variants, identified from approximately 75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of >/=0.1 min(-1), and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences and Department of Chemistry, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Many sequence variations of the 8-17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8-17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5' rN(18)-N(1.1) 3') cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3' position (N(1.1)) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5' position (rN(18)) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8-17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8-17 variants, identified from approximately 75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of >/=0.1 min(-1), and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

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Cleavage versatility of the WT 8–17 sequence. (A) Autoradiogram demonstrating cleavage of all 16 dinucleotide junctions. 5′-32P-labelled substrate was incubated with (+) or without (−) WT for a designated period of time. Reaction buffer: 400 mM KCl, 100 mM NaCl, 7.5 mM MgCl2, 7.5 mM MnCl2 and 50 mM HEPES pH 7.0 at 23°C. Cleavage products were separated from uncleaved substrate by 10% denaturing PAGE. Dinucleotide junctions are organized into related groups, and written 5′-3′ as rN18N1.1. (B) Histogram of rate constants versus dinucleotide junction. kobs values represent the average of at least two independent trials, which typically differed by <30%. Background rate of cleavage under reaction conditions is ∼10−7 min−1.
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Figure 2: Cleavage versatility of the WT 8–17 sequence. (A) Autoradiogram demonstrating cleavage of all 16 dinucleotide junctions. 5′-32P-labelled substrate was incubated with (+) or without (−) WT for a designated period of time. Reaction buffer: 400 mM KCl, 100 mM NaCl, 7.5 mM MgCl2, 7.5 mM MnCl2 and 50 mM HEPES pH 7.0 at 23°C. Cleavage products were separated from uncleaved substrate by 10% denaturing PAGE. Dinucleotide junctions are organized into related groups, and written 5′-3′ as rN18N1.1. (B) Histogram of rate constants versus dinucleotide junction. kobs values represent the average of at least two independent trials, which typically differed by <30%. Background rate of cleavage under reaction conditions is ∼10−7 min−1.

Mentions: The cleavage site versatility of the WT 8–17 sequence is illustrated in Figure 2. Remarkably, all 16 dinucleotide junctions were in fact susceptible to cleavage. Rate enhancements varied by several orders of magnitude, from as low as ∼40-fold (UT junction) to as high as ∼5 × 107 fold (GG junction) over background. As a group, the NG dinucleotide junctions were the most susceptible to cleavage, with rates ranging from ∼0.1 to 5 min−1. The remaining NA, NC and NT junctions were at least two orders of magnitude less reactive, with rates that did not exceed ∼0.001 min−1. Within each group of dinucleotide junctions defined by the same 3′-nt (i.e. the NG group, NA group, NC group and NT group), the reactivity consistently followed the order N = G > A > C > U.Figure 2.


Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme.

Schlosser K, Gu J, Sule L, Li Y - Nucleic Acids Res. (2008)

Cleavage versatility of the WT 8–17 sequence. (A) Autoradiogram demonstrating cleavage of all 16 dinucleotide junctions. 5′-32P-labelled substrate was incubated with (+) or without (−) WT for a designated period of time. Reaction buffer: 400 mM KCl, 100 mM NaCl, 7.5 mM MgCl2, 7.5 mM MnCl2 and 50 mM HEPES pH 7.0 at 23°C. Cleavage products were separated from uncleaved substrate by 10% denaturing PAGE. Dinucleotide junctions are organized into related groups, and written 5′-3′ as rN18N1.1. (B) Histogram of rate constants versus dinucleotide junction. kobs values represent the average of at least two independent trials, which typically differed by <30%. Background rate of cleavage under reaction conditions is ∼10−7 min−1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Cleavage versatility of the WT 8–17 sequence. (A) Autoradiogram demonstrating cleavage of all 16 dinucleotide junctions. 5′-32P-labelled substrate was incubated with (+) or without (−) WT for a designated period of time. Reaction buffer: 400 mM KCl, 100 mM NaCl, 7.5 mM MgCl2, 7.5 mM MnCl2 and 50 mM HEPES pH 7.0 at 23°C. Cleavage products were separated from uncleaved substrate by 10% denaturing PAGE. Dinucleotide junctions are organized into related groups, and written 5′-3′ as rN18N1.1. (B) Histogram of rate constants versus dinucleotide junction. kobs values represent the average of at least two independent trials, which typically differed by <30%. Background rate of cleavage under reaction conditions is ∼10−7 min−1.
Mentions: The cleavage site versatility of the WT 8–17 sequence is illustrated in Figure 2. Remarkably, all 16 dinucleotide junctions were in fact susceptible to cleavage. Rate enhancements varied by several orders of magnitude, from as low as ∼40-fold (UT junction) to as high as ∼5 × 107 fold (GG junction) over background. As a group, the NG dinucleotide junctions were the most susceptible to cleavage, with rates ranging from ∼0.1 to 5 min−1. The remaining NA, NC and NT junctions were at least two orders of magnitude less reactive, with rates that did not exceed ∼0.001 min−1. Within each group of dinucleotide junctions defined by the same 3′-nt (i.e. the NG group, NA group, NC group and NT group), the reactivity consistently followed the order N = G > A > C > U.Figure 2.

Bottom Line: In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8-17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions.We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5' rN(18)-N(1.1) 3') cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3' position (N(1.1)) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5' position (rN(18)) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity.Three optimal 8-17 variants, identified from approximately 75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of >/=0.1 min(-1), and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences and Department of Chemistry, McMaster University, Hamilton, Ontario, Canada.

ABSTRACT
Many sequence variations of the 8-17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8-17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5' rN(18)-N(1.1) 3') cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3' position (N(1.1)) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5' position (rN(18)) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8-17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8-17 variants, identified from approximately 75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of >/=0.1 min(-1), and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.

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