Modulation of the HRAS1 I‑motif DNA Promoter Region by Dietary Plant Flavonoid Kaempferol

Abstract

I-motif (IM) noncanonical DNA structures exist in cellular environments and are implicated in various diseases, including aging, cancer, and neurological disorders, making them attractive targets for drug development. IM DNA can act as molecular switches regulating transcription, especially within oncogenic promoter and telomere regions. This study investigates the interaction between kaempferol (KAE), a dietary flavonoid, with multiple IM DNA configurations alongside duplex DNA using extensive spectroscopic and thermodynamic approaches. UV-vis absorption studies show a 26.67% hyperchromic effect and a notable 15 nm red shift in the absorbance maxima of KAE upon HRAS1 IM binding. Fluorescence measurements indicate a 3.7-fold increase in emission intensity and a 17 nm red shift, coupled with an approximate 3 °C increase in melting temperature (T _m), reflecting strong stabilization of HRAS1 IM by KAE. Our analyses also reveal that KAE preferentially binds HRAS1 IM with a binding constant (K _b) of approximately 4 × 10⁴ M^-1, significantly greater than that of duplex DNA (0.41 × 10⁴ M^-1). Time-resolved fluorescence decay and circular dichroism data support a predominant strong stacking mode of interaction. Thermodynamic parameters suggest that the binding process is spontaneous and enthalpy-driven. These findings demonstrate the potential of KAE to selectively target and stabilize HRAS1 IM DNA, suggesting a promising strategy for transcriptional regulation and anticancer drug design. Furthermore, KAE's pronounced excited-state intramolecular proton transfer fluorescence properties may serve as innovative tools for detecting and quantifying HRAS1 IM DNA in tumor cells, with potential applications in theranostics.

1

Schematic representation of basic structures of flavonoid [a] and kaempferol (KAE) [b].

2

UV–vis absorption spectra of KAE (15 μM) in the absence and presence of successive additions of HRAS IM [a] & Duplex DNA [b] (30 μM). (IM buffer, pH 5.4, Duplex buffer, pH 7.4, temp. 25 °C).

3

Photoinduced excited state intramolecular proton transfer (ESIPT) in KAE. Ground and excited (indicated by *) states of normal (N) and tautomer (T) forms of KAE.

4

Fluorescence emission spectra of 10 μM KAE (λ_ex = 365 nm) in the presence of increasing concentrations of HRAS1 IM [a] & Duplex DNA [b] (30 μM). (IM buffer, pH 5.4, Duplex buffer, pH 7.4, temp. 25 °C).

5

Modified Benesi–Hildebrand double reciprocal plots for determination of binding constant between KAE with HRAS1 IM [a] & Duplex DNA [b]. (IM buffer, pH-5.4, duplex buffer, pH 7.4, temp. 25 °C).

6

Fluorescence anisotropy (r) for the KAE (10 μM, λ_ex = 365 nm) in experimental buffer and also in the HRAS1 IM [a] & Duplex DNA [b] (10 μM & 20 μM). (IM buffer, pH-5.4, Duplex buffer, pH-7.4, temp. 25 °C).

7

Time-resolved fluorescence decay profile of KAE (10 μM, λ_ex = 365 nm) in experimental buffer and in the presence of HRAS1 IM [a] & Duplex DNA [b], Δτ_avg = 2.16 ns & Duplex DNA [b], Δτ_avg = 0.2 ns (10 μM & 20 μM) (IM buffer, pH-5.4, Duplex buffer, pH-7.4, temp. 25 °C).

8

Circular dichroic spectral profile of HRAS1 IM [a] & Duplex DNA [b] (20 μM) in the presence of KAE (20, 40, & 60 μM). (IM buffer, pH 5.4, Duplex buffer, pH 7.4, temp. 25 °C).

9

UV melting profiles of HRAS1 IM [a] & Duplex DNA [b] (10 μM) alone and in the presence of KAE (20 μM). Variations of the IM DNA melting temperature (ΔT _m) in °C were induced by KAE. (IM buffer, pH 5.4, Duplex buffer, pH 7.4, temp. 25 °C).

10

Fourier transform infrared (FT-IR) spectra in the [800–2500] cm^–1 spectral region of HRAS1 IM (a), duplex DNA (b) (black line), and HRAS1 IM + KAE (a), Duplex + KAE (b) (red line). The peak center of each spectrum is indicated by the arrows (IM buffer, pH 5.4, Duplex buffer, pH 7.4, temp. 25 °C).