Researchers Make Progress Towards Engineering Restriction Enzymes

Restriction Enzymes

Researchers at Mount Sinai Medical have recently made progress towards engineering restriction enzymes (RM systems) with specified recognition sequences using the enzyme MmeI. These enzymes are widely used in genome editing and cloning.

Biotechnology, An Advancing Field

The isolation and manipulation of molecular components has steadily improved since the late 1950’s and early 1960’s when scientists discovered and began to characterize DNA [1]. Since then, science has produced technology that can control and quantify biological systems to unprecedented levels. This has allowed scientists not just sequence DNA but also completely alter an organism’s genetic code. In order to engineer better biological tools, molecular biologists have worked to alter natural molecules to suit their purposes. One example of such molecules are restriction endonucleases (REases). These restriction enzymes cut DNA at specific points relative to a specific recognition sequence (called “restriction sites”). Restriction endonucleases have been widely used in genome editing, cloning, and DNA fingerprinting. Recently, work has shifted towards understanding how REases function so that new or modified versions can be designed.

Restriction Enzymes

Restriction endonucleases are used in conjunction with methyltransferases (MTases) to produce a restriction enzyme (RM system). The REase will cut DNA while the methyltransferase protects DNA nearby through the addition of a methyl group (methylation) [3]. One example of how this system is used in nature can be observed in bacteria protecting themselves from viral DNA. MTase will methylate the parts of DNA near the restriction site on the host DNA so that the restriction endonuclease will only cut up the invading viral DNA [4].

Type II REases are widely used in modern biotechnology. Although over 4,000 type II REases have been discovered,  many of them use the same restriction sites. In total, there are only about 365 different known restriction site recognized be REases [5, 6]. This has led to a desire in the scientific community to be able to create REases that can be programed to recognize any sequence of DNA.

Engineering New Selectivity

Researchers at Mount Sinai Medical have recently made progress towards engineering restriction enzymes (RM systems) with specified recognition sequences using the enzyme MmeI [6]. MmeI comes from the bacteria Methylophilus methylotrophus and is used to protect the bacteria from foreign DNA. MmeI is particularly interesting because it has a relatively large distance between the restriction site and the cleavage site. This makes it particularly useful for some next generation DNA sequencing applications including serial analysis of gene expression (SAGE) and paired-end tags (PET).  MmeI was chosen by the group of researchers because it is bifunctional, meaning the REase and the MTase share the same target recognition domain (TRD). Because of this, the RM system has more capability to change its specificity to combat evolving viruses. This makes the RM system easily mutable: as the sequence recognized by the enzyme can be altered by making small changes to the amino acid sequence of the RM enzyme [8, 9].

After analyzing the structure of MmeI, the group was able to determine the amino acids that play a role in DNA sequence recognition, they also were able to find clues as to how MmeI is able to cleave so far from the restriction site, an attribute they referred to as “long reach”. They were successfully able to alter the 3rd, 4th, and 6th nucleotides in the recognition sequence, however, they have not yet fully overcome the immutability of the 1st, 2nd, and 5th position nucleotides [6].


The work of these researchers shows the continued effort to create RM systems engineered with unique specificities. Achieving this will expand the uses of RM systems allowing them to be used in more specific circumstances. Instead of designing experiments around the available recognition sequences, RM systems will eventually be custom designed for individual applications. Molecular biology is on the precipice of breaking away from many constraints brought about from only having access to components and systems that are already in use in nature. Systems Engineering of biological systems will benefit greatly from being able to not just characterize parts, but produce novel parts as well.



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    By: Keegan Curry

    Keegan Curry is an undergraduate bioengineering student at the University of Louisville- J.B. Speed School of Engineering. After getting his bachelor’s degree he will pursue a Master of Engineering in bioengineering. Keegan does research with biopolymers and drug delivery at the Clinical and Translational Sciences Institute.

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