Question

Question 4.

Describe an experiment to demonstrate whether the RNA folds into a functional tertiary structure of a ribozyme when the tetraloop region is deleted. You suspect that the tetraloop is critical for folding of a ribozyme into its active form.

Answer 1

The rapid and gold standard experiment is by setting up Elisa based Ribozyme kinetic assay ( Kossen et al, Chemistry & Biology, Vol. 11, 807–815, June, 2004)

The schematic view of the assay is given in the above diagram.

1) 96 well plate base

2) Streptavidin coating

3) Bioting tag 3' end of RNA under study

4) Tertiary stcuture of RNA

5) 5' Fluorescien tag ( 5' end of RNA under study)

6) Alkaline phosphatase enzyme tagged Antobody which is specefic to Flurescein end of RNA tertiary form

7) Substrate for Alkaline phosphatase

8) Product ( fluorescence)

Principle of the Assay: If the RNA under study contains the tetraloop region, it folds in the suitable condition.The folding and formation of tertiary stucture by RNA results in the activation of 5' Fluorescein tag to achieve its active form. This active form is detected by the specefic antibody tagged with Alkaline phosphatase enzyme. When substrate is added , the enzyme coverts it into fluorescent product which can be used as signal.

In order totrace the tetraloop region that is critical for attaining the tertiry structure , we may perform mutagenesis and replace the tetraloop region nucleosides. The Elisa based assay described above can be used to determine the critical tertraloop region within the RNA. If the traloop region is mutated , then RNA will not attain the teritiary form, hence the fluorescent signal will not appear.