Team:UANL Mty-Mexico/project/DNA-Program-Supression

DNA Specific Deletion

The DNA, just like a lot of other molecules, suffers from deletion; be it to repair, insert a fragment, recombine or as a defense strategy. There are multiple causes for this, but the one that interest us is due to enzymatic action, among which we find exonucleases, restriction enzymes, and other molecules that will be revised shortly.

Endonucleases

Nucleases are enzymes with the ability to fragment DNA through phosphodiester bond ruptures. When the cutting site is in the 5’ or 3' end, it’s called an exonuclease; on the other hand, if it's inside the DNA strand, it´s called an endonuclease. Among endonucleases, restriction enzymes, which can recognize specific DNA sequences (Sui-Hong et al, 2010), have been of utmost interest in the manipulation of DNA, from polymorphism identification (molecular diagnosis) to the construction of new DNA sequences (genetic engineering).

According to their characteristics, they are divided into four types, from I to IV. Moreover, each of them has their own specific applications. The most studied and used are type II restriction enzymes, because they recognize a specific palindromic sequence and they generally inside this sequence. On the other hand, type I enzymes cut approximately at a 1000 bp distance, while type III cuts at a 24-26 bp distance. Finally, type IV has low specificity and only cuts methylated DNA (Roberts et al, 2003).

Among type 2 restriction enzymes, there are those who can cut just one strand, which is called nicking. They are generally named with an N prefix, for example, N.bstSEI (Roberts et al, 2003).

Restriction enzymes can recognize symmetric and asymmetric sequences (Pingoud et al, 2001). One way of classifying those that recognize asymmetric sequences is in 5 classes according to their characteristics (Sui-Hong et al, 2010), which are shown in the table.

Type II restriction enzymes have come to be used so much in the molecular biology field, that commercially they are the most exploited. But even among them, there can be advantages and disadvantages. For example, if an enzyme has a recognition site of a few nucleotides, it is better suited for diagnostic trials than for genetic engineering. For this reason the use and research of new enzymes has begun, along with the de novo design of others (Sui-Hong et al, 2010).

The mode of action of type II restrictions enzymes can be imitated with artificially designed enzymes. For example, the union of proteins and/or peptides that recognize certain sequences with others that have the capacity of excising DNA.

In our project we plan to use endonucleases, together with polypeptides that recognize longer sequences than restriction enzymes, in order to increase the specificity.

Zinc- Finger Nucleases (ZFN)

Zinc-finger nucleases are agents that have been used for DNA modification by means of the fusion of a zinc finger, designed or preexistent, with the active domain of the Fokl enzyme; this Phusion molecule is called “Zinc Finger Nuclease” or ZFN (Kim et al, 1996). They have been used in different organisms, from animals to plants (Miller et al, 2007) with the purpose of modifying them, for example, through an integration of complete genes (Moehle et al, 2007).

An important aspect to be considered before using this technology, is that in order to cut it needs to dimerize with the functional domain of another Fokl (Bitinaite, 1998). That’s why it´s required to design adjacent ZFNs whose Fokl domains interact in an intermediate site. There are also other advantages, like its high specificity due to the ability to design binding sites of over 18 bp (Urnov et al, 2005) and because they normally only cut once they have bound to the specific site (Vanamee et al, 2001). On the other hand, when the ZFP has not joined its specific site, Fokl remains as a monomer even to the 15 µM (Kaczorowski et al, 1989), making the appearance of cuts in non-specific sites more difficult.

However, it is necessary to mention that some possible disadvantages exist, ones that may surface in spite of good planning. One of them is that the effectiveness of a ZFN in one species doesn’t it will function in others (--). Another downside may be that the designed ZFNs work, but once they homodimerize they cut sites they weren’t designed for, becoming toxic for the cell (Beumer et al, 2006). Due to this, the designing of new ZFN requires experimentation to assure its correct functioning.

In our project ZFNs will be used, and to avoid the problems mentioned above, we will use ZFNs that have been previously tested in E. coli. This is because our aim is to prove the utility of said protein in our project.

CRISPR Cas9 (Adaptative immune system)

Characteristics:
1. System guided by RNA
2. Method that controls genetic expression
3. Cas9 endonuclease