Enzymatic Cleavage of Type II Restriction Endonucleases on the 2′-O-Methyl Nucleotide and Phosphorothioate Substituted DNA

Abstract

The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2′-O-methyl nucleotide (2′-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2′-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.

Production of dumbbell probe through hairpin cleavage-ligation and increasing RCA sensitivity and specificity by circle to circle amplification

“…An optimum analog should resist RE cleavage without interfering with polymerization reaction of RCA. From a selecting study, we found phosphorothioate (PS) modification at A5pG6 position can strongly suppress PstI activity, and PS had little effects on RCA efficiency 33 34 . Consequently, we used DP with a PS-modified PstI hexamer in the stem to conduct this coreaction.…”

Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution

“…These enzymes are commonly used for cloning and plasmid linearization. Because the evolution of enzymes with custom restriction endonuclease activity is an attractive area of research, we adapted an optical sensor that was previously developed for use in bulk solution . Accordingly, the sensor consists of a nicked duplex in which enzymatic activity disrupts a fluorophore-quencher pair adjacent to the digestion site (Figure c).…”

Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases

“…2′-F RNA and LNA locks sugar ring in C3′ endopucker conformation and has many advantages such as increasing affinity to RNA target 5,37) and protecting oligonucleotide against nuclease digestion 38) . In our previous study, we also found their applications in regulating endonuclease cleavage efficiency 39,40) . Though 2′-OMeRNA has similar characteristics of the C3′ endopucker conformation, it should be avoided in RCA application since it strongly suppressed RCA efficiency.…”