In #biologics development, a lot of success depends on vector designing/ engineering as only an unique combination of the elements yields the best results. One of these vector elements are S/MARs (Scaffold/ Matrix Attachment Regions).
Introduction:
The nuclear matrix is a 3-D filamentous #RNA–protein meshwork. The chromatin is organized into loops by virtue of #DNA sequences that tether the chromatin to the nuclear matrix. These anchor sequences are known as S/MARs. S/MAR binding proteins (S/MARBPs) interact with S/MARs to facilitate chromatin looping which in turn helps in many cellular processes like DNA #replication, #transcription, #recombination, chromosome #condensation, chromatin to chromosome transition and #DNArepair.
Mechanism of action:
S/MARs separate a given #transcriptional unit from its neighbors and also provide platforms for the assembly of factors enabling transcriptional events within the unit. These AT-rich regions potentially give rise to a bending nature in DNA sequences that allows the groove to facilitate the binding of DNA-binding proteins.
An increased propensity to separate the DNA strands can serve the formation of secondary structures such as #cruciforms or slippage structures, which are recognizable features for a number of enzymes like #DNases, #topoisomerases, poly(ADP-ribosyl) polymerases and enzymes of the histone-acetylation and DNA-methylation apparatus.
Also, #topoisomerase IIA cleavage sites have been found to be frequently associated with S/MARs, because topoisomerase IIA can resolve topological problems caused by knotting and supercoiling at S/MARs. Therefore, AT-rich regions cleaved by topoisomerase IIA might facilitate #DSBs that in turn can be utilized for transgene integration.
Characteristics:
MARs at different locations in plasmid elicit variable effects thereby suggesting a position effect.
Insertion orientation of MARs has no significant effect on transgene incorporation into chromosome.
Applications:
In one of the papers scientists used human MAR X-29 (GenBank No. EF694970.1, Size: 3.3kb) in a vector as (MAR-Spacer-CMV promoter-Gene-PolyA). Here, with a spacer of 500bp, gene copy number in host chromosome increased 1.77 fold than control vector without MAR, and with spacer of 1000bp there was a 1.56 fold increase in copy number.
In a study by Ley et al. (2013), scientists have shown that MAR X-29-containing transposons mediate elevated expression of therapeutic proteins in polyclonal or monoclonal #CHO cell populations using a transposable vector devoid of selection gene.
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