Page 54 - Annual report 2021-22

Annual Report 2021-22 |

Reverse probing of the Rab11 binding to CHAST was carried out. For this, anti-Rab11 antibody was
procured and pull-down with subsequent qPCR was performed. CHAST was found to be enriched in
this fraction. Binding studies were performed after purification and establishing the quality of all three
proteins. Rab11, alpha actinin-1 and lamin were tested for their binding ability towards the quadruplex
forming CHAST oligo-1 using electrophoretic mobility shift assays (EMSA). Rab11 showed strong
binding with the oligo-1, however alpha actinin-1 and lamin did not show binding upto a concentration 37
of 20 µM. It should be noted that alpha actinin-1 and lamin may bind to other secondary structures in
CHAST (mainly the hairpin) or bind to the quadruplex region with lower affinity.

The structure of CHAST was analyzed using RNAalifold and minimal regions that may be sufficient for
activity have been optimized (truncated regions synthesized through in-vitro transcription). Mainly,
the sequence of CHAST was tiled and overlapping sequences containing hairpin loops and putative
quadruplex regions were identified and optimized. Their interaction with different partner proteins is
currently under progress. The in vitro transcribed products were biotinylated and purified using
column purification and purified constructs were subjected to pull-down experiments to identify a
sub-set of protein binding partners. This experiment is currently being optimized. Differences in their
thermodynamic parameters in comparison to the full-length CHAST will then be compared using
biochemical and biophysical methods.

“Chemistry must become the astronomy of the molecular world.”
— Alfred Werner