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. 2021 Jul 19;6(30):19757-19770.
doi: 10.1021/acsomega.1c02451. eCollection 2021 Aug 3.

Molecular Assembly of Triplex of Duplexes from Homothyminyl-Homocytosinyl Cγ(S/ R)-Bimodal Peptide Nucleic Acids with dA8/dG6 and the Cell Permeability of Bimodal Peptide Nucleic Acids

Pramod Bhingardeve  1 , Prashant Jain  1 , Krishna N Ganesh  1   2
Affiliations

Molecular Assembly of Triplex of Duplexes from Homothyminyl-Homocytosinyl Cγ(S/ R)-Bimodal Peptide Nucleic Acids with dA8/dG6 and the Cell Permeability of Bimodal Peptide Nucleic Acids

Pramod Bhingardeve et al. ACS Omega. .

Abstract

Peptide nucleic acids (PNAs) are analogues of DNA with a neutral acyclic polyamide backbone containing nucleobases attached through a t-amide link on repeating units of aminoethylglycine (aeg). They bind to complementary DNA or RNA in a sequence-specific manner to form duplexes with higher stablity than DNA:DNA and DNA:RNA hybrids. We have recently explored a new type of PNA termed bimodal PNA (bm-PNA) designed with two nucleobases per aeg repeating unit of PNA oligomer and attached at Cα or Cγ of each aeg unit through a spacer sidechain. We demonstrated that Cγ-bimodal PNA oligomers with mixed nucleobase sequences bind concurrently two different complementary DNAs, forming double duplexes, one from each t-amide and Cγ face, sharing a common PNA backbone. In such bm-PNA:DNA ternary complexes, the two duplexes show higher thermal stability than individual duplexes. Herein, we show that Cγ(S/R)-bimodal PNAs with homothymines (T8) on a t-amide face and homocytosine (C6) on a Cγ-face form a conjoined pentameric complex consisting of a triplex (bm-PNA-T8)2:dA8 and two duplexes of bm-PNA-C6:dG6. The pentameric complex [dG6:Cγ(S/R)-bm-PNA:dA8:Cγ(S/R)-bm-PNA:dG6] exhibits higher thermal stability than the individual triplex and duplex, with Cγ(S)-bm-PNA complexes being more stable than Cγ(R)-bm-PNA complexes. The conjoined duplexes of Cγ-bimodal PNAs can be used to generate novel higher-order assemblies with DNA and RNA. The Cγ(S/R)-bimodal PNAs are shown to enter MCF7 and NIH 3T3 cells and exhibit low toxicity to cells.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(a) aeg-PNA, (b) DNA, (c) Cα(R)-bm-triazole-PNA, (d) Cγ(S)-bm-PNA, (e) Cγ(R)-bm-PNA, (f) Cα(S)-bm-PNA:DNA double duplex, and (g) Cγ(S/R)-bm-PNA:DNA double duplex. B = T/A/G/C.
Figure 2
Figure 2
H-bonded pentameric triplex of duplexes that constituted from homothyminyl-homocysteinyl Cγ(S/R)-bm-PNA and DNA strands. Strands 1, 3, and 5 are DNA, and strands 2 and 4 are bimodal PNA.
Figure 3
Figure 3
Structures of Cγ(S/R)-bm-PNAs (1 and 2) and Cγ(S/R)-iso-PNAs (3 and 4).
Figure 4
Figure 4
Structures of protected monomers used in the solid-phase synthesis of PNA oligomers. 1. Cγ (S)-bm-PNA-T; 2. Cγ(R)-bm-PNA-T; 3. Cγ(S)-iso-PNA-T; 4. Cγ(R)-iso-PNA-T. eam, ethylamino; aeg, aminoethylglycyl.
Scheme 1
Scheme 1. Common Protocol for Synthesis of Cγ(S/R)-bm-PNA Oligomers from Monomers 1 and 2 and Cγ(S/R)-iso-PNA Oligomers from Monomers 3 and 4
The wavy line represents the Cγ sidechain with S or R stereochemistry. Typical coupling reaction: microwave, 25 W; 5 min; rt, 6 h. Resin, 25 mg (∼0.2 mmol/g); monomers (12 mg, 3 equiv): (a) (i) HOBt (3 mg, 3 equiv), HBTU (6 mg, 3 equiv), DIPEA (6 μL), DMF. (b) (i) 50% TFA in DCM; (ii) neutralization with 10% DIPEA in DCM. (c) 20% piperidine in DMF. (d) (ii) TFA-TFMSA, thioanisole.
Figure 5
Figure 5
Structure and UV–T plots of Cγ-duplexes of Cγ(S/R)-iso/bm-PNA with DNA 1 (dG6). (A) Cγ(S)-iso-PNA 3:dG6; (B) Cγ(R)-iso-PNA 4:dG6; (C) Cγ(S)-bm-PNA 1:dG6; (D) Cγ(R)-bm-PNA 2:dG6 . Red curve, melting curve; blue dotted curve, first derivative plot. Numbers in figures indicate Tm’s. Sodium cacodylate (10 mM), NaCl (10 mM), pH 7.2.
Figure 6
Figure 6
Structure and UV–T plots of Cγ-triplexes of Cγ(S/R)-iso/bm-PNA with DNA 2 (dA8). (A) [Cγ(S)-bm-PNA-T 3]2:dG6 and (B) [Cγ(R)-bm-PNA-T 4]2:dG6. Red curve, melting curve; blue dotted curve, first derivative plot. Numbers in figures indicate Tm’s. Sodium cacodylate (10 mM), NaCl (10 mM), pH 7.2.
Figure 7
Figure 7
Structures of pentameric triplex of duplexes and UV–T plots of Cγ(S/R)-bm-PNA:DNA 1:DNA 2 complexes. (A) dG6:Cγ(S)-bm-PNA 1:dA8:Cγ(S)-bm-PNA 1:dG6. (B) dG6:Cγ(R)-bm-PNA 2:dA8:Cγ(R)-bm-PNA 2:dG6. Buffer: sodium cacodylate (10 mM), NaCl (10 mM), pH 7.2.
Figure 8
Figure 8
Comparative (A) Tm’s of duplexes, triplexes, and triplex of duplexes and (B) ΔTm of mismatch complexes. Labels on the x axis correspond to complexes shown in Figures 5 and 7 and this figure. The numbers in panel (B) indicate the destabilization (ΔTm) of mismatched duplexes in °C.
Figure 9
Figure 9
CD spectra of Cγ(S/R)-bm-PNA:DNA complexes. 5C duplex: dCγ(S)-bm-PNA 1:dG6; 6A triplex: [Cγ(S)-bm-PNA 1]2:dA8; 7A triplex of duplex: dG6:Cγ(S)-bm-PNA 1:dA8:Cγ(S)-bm-PNA 1:dG6. 5D, 6B, and 7B are the corresponding complexes of Cγ(R)-bm-PNA 1. Structures of complexes are as shown in Figures 5–7. Sodium cacodylate (10 mM), NaCl (10 mM), pH 7.2.
Figure 10
Figure 10
(A) Schematic representation of stepwise addition of DNA 1 and DNA 2 to Cγ(S/R)-bm-PNA by two paths. Path I: Triplex formation after step a followed by its duplex formation with dG6. Path II: Duplex formation with dG6 in step c followed by its triplexation with dA8 to yield a triplex of duplex. (B, C) CD spectra of products; curve a: triplex 6A after step a; curve b, triplex of duplex 7A after step b; curve c, duplex 5C after step c; curve d, triplex of duplex 7A after step d. (D) Overlap of CD spectra of compounds in paths I (red) and II (blue). Sodium cacodylate (10 mM), NaCl (10 mM), pH 7.2.
Figure 11
Figure 11
Cell uptake confocal fluorescence images of bm-PNA, iso-PNA, and aeg-PNA in MCF7 cells (A) DAPI-stained and (B) treated with Cf-PNA and NIH 3T3 cells (C) DAPI-stained and (D) treated with Cf-PNAs. The individual PNAs are shown on top of each column of panels.
Figure 12
Figure 12
Percentage cell uptake of aeg-PNA, Cγ(S/R)-iso-PNAs, and Cγ(S/R)-bm-PNAs measured from FACS in (A) MCF7 and (B) NIH 3T3 cells.
Figure 13
Figure 13
Cell viability measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for (A) MCF7 cells and (B) NIH 3T3 cells after treatment with 4 μM bimodal PNAs (Cγ(S)-bm-PNA 1 and Cγ(R)-bm-PNA 2), iso-PNA (Cγ(S)-iso-PNA 3 and Cγ(R)-iso-PNA 4), and aeg-PNA (aeg-PNA-C65 and aeg-PNA-T86) for 12 h. The data shown are the average of three measurements. The error bars represent standard deviations. Control experiments do not have any added PNAs.
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