Antisense and Ribozyme Technology
Among several possible strategies for inactivating a single, chosen gene the most approved one is Antisense technology. This modification does not involve actual subtraction but inactivation of gene or suppressing gene activity. When DNA strand is normally transcribed the RNA produced is called sense RNA, complementary to DNA but if orientation of gene to be transcribed is reversed with respect to promoter the RNA transcribed from it would be reversed too, the RNA produced so has sequence same to that of antisense strand of the normal gene, thus known as Antisense RNA (asRNA).
The Basis of Antisense Technology is the use of antisense RNA, the gene which is to be cloned is ligated into the vector in reverse orientation thus on transcription the RNA synthesized is reverse complement of the mRNA transcribed from normal version of gene and being complementary to each other they will pair to form hybrid.
* Makes mRNA unavailable for translation,
* Resulting double stranded RNA molecule is degraded by specific Ribonucleases.
The application of Antisense technology is seen most in plant genetic engineering.
1) Slow Ripening Tomato: In Tomato in the later stages of ripening polygalactouronase gene is switched on coding for polygalactouronase enzyme which breaks down polygalactouronic acid component of cell walls resulting in softening which results in spoilt tomato. Transgenic tomatoes were prepared containing antisense construct of the gene PG resulting in reduced expression of PG and slow ripening and fruit softening, improving shelf life.
2) Inactivate ethylene synthesis
3) Modification of flower color in decorative plants
More application will undoubtedly follow in future.
Stating that all enzymes are proteins is wrong. In 1990, Tom Cech and Sidney Altman shared the Nobel Prize for their demonstration that RNA could act as an enzyme. �Ribozymes� are antisense RNA molecules with enzymatic properties, or RNAs that, like those in ribonuclease P, make up part of an enzyme that contains other compounds as well. They function by binding to the target RNA moiety through base pairing and inactivate it by cleaving the phosphodiester backbone at a specific cutting side. The various types of reactions performed by ribozymes are based on transesterification; these reactions include
Splicing, oligonucleotide chain extension, RNA ligation, endonuclease action and phosphatase action. The ribozyme action is generated by formation of particular secondary and tertiary structures that create active sites. A ribozyme has two sites: a substrate binding site and a guanosine-binding site.
Five classes of ribozymes have been described based on sequences as well as three-dimensional structures. They are:
1) Tetrahymena group I intron
2) Rnase P
3) Hammerhead ribozyme
4) Hairpin ribozyme
5) Hepatitis delta virus ribozyme
Ribozymes have the potential to become useful therapeutic agents and currently the vast majority of effort has been expended in the development of trans cleaving hammerhead and hairpin ribozymes as inhibitors of viral gene expression, in particular to cleave and destroy HIV-I RNAs to inhibit viral replication in infected cells. RNA enzymes can potentially be quite useful for a variety of gene therapy applications.