Targeting a KRAS i-motif forming sequence by unmodified and gamma-modified peptide nucleic acid oligomers

Abstract

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

Keywords: KRAS gene; gamma PNA; hybridization; i-motif; peptide nucleic acid; strand invasion.

FIGURE 1

(a) Chemical structure of peptide nucleic acid (PNA) and γ‐modified PNA (γPNA) monomers. (b) Generic i‐motif structure, stabilized by C:C⁺ base pairing. (c) Targeting i‐motif DNA derived from far‐region of KRAS promoter by complementary γPNA

CHART 1

Sequences of DNA, unmodified and γ‐modified PNA oligomers used in this study. Cytosine tracts are highlighted in yellow. P1, γP1 are complementary to underlined nucleotides. (PNA/γPNA N‐terminus aligns with the DNA 3′‐terminus.) Two l‐lysine residues were added to the C‐terminus of the purine‐rich PNA probes to improve solubility; these were also included in the γPNA probes for consistency. PNA and γPNA residues written as lower and upper case, respectively

SCHEME 1

Hybridization of PNA (P1) or γPNA (γP1) to i‐motif forming sequence D41 will produce heteroduplexes. C‐tracts involved in i‐motif formation are highlighted in yellow.

FIGURE 2

(a) CD spectra recorded at 37°C, (b) UV melting at 260 nm and (c) CD melting at 285 nm for 1 μM D41 at pH 5 (solid lines) and pH 7.4 (dashed lines). CD spectra were collected after heating the samples at 95°C for five mins before cooling to 37°C. For both UV and CD melting, the heating ramp is shown and was recorded at the rate of 1°C/min

FIGURE 3

(a) Fluorescence spectra of 2 μM QR with (red) or without (black) 0.2 μM D41 recorded at 37°C in potassium phosphate buffer of pH either 5 (solid line curves) or 7.4 (dashed curves). (b) The average fluorescence intensity of 2 μM QR + 0.2 μM D41 at 628 nm wavelength recorded at different temperatures in potassium phosphate buffer of pH either 5 (black circles) or 7.4 (red circles). λ _ex = 550 nm

FIGURE 4

CD spectra of 1 μM P1 (dashed lines) and γP1 (solid lines) recorded at 37°C at (a) pH 5 and (b) pH 7.4

FIGURE 5

CD spectra of 1 μM D41 alone (dashed lines) and with equimolar P1/γP1 (solid lines) recorded at 37°C at pH 5 (a and c) and pH 7.4 (b and d)

FIGURE 6

CD melting curves recorded at 260 nm for 1 mM D41‐P1 (dashed lines) and D41‐γP1 (solid lines) recorded at pH 5 (a) and pH 7.4 (b). In each case, the heating ramp is shown and was recorded at the rate of 1°C/min

SCHEME 2

Proposed mechanism for formation of intermolecular i‐motif in response to PNA/γPNA hybridization to D41. I‐motif invasion (Step 1) leads to heteroduplex formation, leaving two C‐tracts available for intermolecular i‐motif formation (Step 2).

FIGURE 7

Fluorescence spectra of QR and QR + D41 in the presence of either P1 or γP1 at (a) pH 5 and (b) pH 7.4 (b). [QR] = 2 μM, [D41] = [P1] = [γP1] = 0.2 μM

FIGURE 8

Control experiments to test for intermolecular i‐motif formation by D41 in the presence of PNA/γPNA. Left: Schematic illustrating hybrids formed with D12, D41 and D41′; right: Normalized fluorescence enhancement relative to free QR. Data and error bars represent mean and standard deviation from three trials

FIGURE 9

Effect of PNA probes P1 and/or P3 on QR fluorescence in the presence of D41 at (a) pH 5 and (b) pH 7.4 (b). [QR] = 2 μM, [D41] = [P1] = [P3] = 0.2 μM

FIGURE 10

Comparison of CD spectra of 1 μM D41 (solid line), D20 (dashed line) and D12 (dotted line) recorded at 37°C at (a) pH 5 and (b) pH 7.4

SCHEME 3

Simultaneous targeting of G‐quadruplex and i‐motif sequences in double‐stranded DNA promoter by γPNA should lead to potent transcription inhibition.