Page 46 - Biennial Report 2018-20 Jun 2021

check for reddening, swelling, blister formations and checked wound healing rate. Further H&E
staining was carried out to decipher morphological changes.

BIOPHYSICAL STUDIES ON RNA-PEPTIDE INTERACTIONS

Non-coding RNAs are functional RNA molecules that get transcribed from DNA, but are not
translated into proteins; yet, they can regulate gene expression at transcriptional and
posttranscriptional levels. Secondary structures present within these RNAs play a major role in
determining their nature of function. In case of miRNAs, the precursor miRNA have hairpin stem
loop structure, which is required for Dicer recognition and further maturation. Structural
transition from hairpin to G-quadruplex depends upon the nucleotide sequence, the cellular
microenvironment and this might affect the miRNA maturation and other downstream activity.

One such structure, G-quadruplex, can modulate the miRNA regulatory role. Formation of G-
quadruplex within precursor miRNA-149 has been shown to inhibit Dicer processing activity
followed by suppression of miRNA-149 maturation and thereby rendering it pathogenic for
cancer. It was shown that suppression of cell proliferation by the upregulated miRNA-149 could
be rescued by unfolding the quadruplex present in pre-miRNA-149 by TmPyP4 (Porphyrin)
treatment. Using UV-visible spectroscopy, circular dichroism and isothermal titration
calorimetry, it was observed that TmPyP4 binds strongly to quadruplex and unfolds it, which was
further verified by NMR Spectroscopy. In-cellulo, qRT-PCR measurements of miRNA-149 in MCF-
7 breast cancer cells showed concentration dependent enhancement of mature miRNA-149
upon treatment of TmPyP4. As a consequence of enhanced miRNA-149 levels, the reduction in
miRNA-149 target protein ZBTB2 could be observed that eventually lead to reduced cell
proliferation.

Besides affecting miRNA biogenesis, quadruplexes also affect telomeres. Telomeric repeat-
containing RNA (TERRA) is a non-coding RNA transcribed from the telomeric regions. In vitro
studies indicated that the TERRA quadruplex interacts with telomeric DNA quadruplex and
controls the telomerase activity. Many proteins that have the capability to interact with G-
quadruplex were studied and proteins were considered significant in targeting the quadruplex
as they are more specific than small molecules. One such protein is RHAU (RNA helicase
associated with AU-rich element), a member of the ATP-dependent DEX (H/D) family of RNA
helicases, with a core helicase domain (470 amino acid) responsible for ATP
binding/hydrolysis/helicase activity and is flanked on either side by N-and C-terminal extensions.
The N-terminal extension is required for quadruplex recognition by resulting in the complex
formation of human telomerase RNA and G-quadruplex. This helicase can bind and unwind the
RNA quadruplex with higher affinity; however, the thermodynamic properties of the complete
interaction remain unknown. Thus, the peptide of 16 amino acid residues of RHAU called HK-16
was chosen for this study. The biophysical and thermodynamic properties of HK-16 interaction
with quadruplex was confirmed by performing EMSA (by NATIVE PAGE), FID assay (Fluorescence
Intercalator Displacement assay), CD (circular dichroism) spectra, CD titrations and ITC
(Isothermal Titration Calorimetry) by Souvik Maiti’s group. The analysis of the thermodynamic
properties concluded that HK-16 interacts with specificity and affinity towards TERRA
quadruplex rather than telomeric DNA quadruplex.

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