The paperclip triplex: understanding the role of apex residues in tight turns

Abstract

In this study, we investigate the role of the apex nucleotides of the two turns found in the intramolecular "paperclip" type triplex DNA formed by 5'-TCTCTCCTCTCTAGAGAG-3'. Our previously published structure calculations show that residues C7-A18 form a hairpin turn via Watson-Crick basepairing and residues T1-C6 bind into the major groove of the hairpin via Hoogsteen basepairing resulting in a broad turn of the T1-T12 5'-pyrimidine section of the DNA. We find that only the C6C7/G18 apex triad (and not the T12A13/T1 apex triad) is required for intramolecular triplex formation, is base independent, and occurs whether the purine section is located at the 5' or 3' end of the sequence. NMR spectroscopy and molecular dynamics simulations are used to investigate a bimolecular complex (which retains only the C6C7/G18 apex) in which a pyrimidine strand 5'- TCTCTCCTCTCT-3' makes a broad fold stabilized by the purine strand 5'-AGAGAG-3' via Watson Crick pairing to the T8-T12 and Hoogsteen basepairing to T1-T5 of the pyrimidine strand. Interestingly, this investigation shows that this 5'-AGAGAG-3' oligo acts as a new kind of triplex forming oligonucleotide, and adds to the growing number of triplex forming oligonucleotides that may prove useful as therapeutic agents.

FIGURE 1

(A) Paperclip configuration of 5′-TCTCTCCTCTCTAGAGAG-3′ (ccag), (B) 5′-CTCTCTTCTCTCGAGAGA-3′ (ttga), (C) 5′-GAGAGATCTCTCCTCTCT-3′ (gacc), (D) 5′-AGAGAGCTCTCTTCTCTC-3′ (agtt), and (E) bimolecular complex of 5′-TCTCTCCTCTCT-3′ (cc) and 5′-AGAGAG-3′ (ag). (|) Indicates Watson Crick basepairing and (*) indicates Hoogsteen basepairing.

FIGURE 2

(A) CD spectra of ccag (solid), ccag-t (dashed), and ccag-g (dotted) at pH 4.5, 25°C. (B) CD spectra of ttga (solid), ttga-c (dashed) and ttga-a (dotted) at pH 4.5, 25°C. (C) CD spectra of gacc (solid), gacc-g (dashed) and gacc-t (dotted) at pH 4.5, 25°C. (D) CD spectra of agtt (solid), agtt-a (dashed) and agtt-c (dotted) at pH 4.5, 25°C. (E) CD spectra of ccag (solid), 6ag +12cc (dashed), and 6ag+12tt (dotted), at pH 4.5, 5°C.

FIGURE 3

Native gel electrophoresis of ag (column I from left), cc (II), cc+ag (III), tt+ag (IV), (AG)₉ (V), and (AG)₁₈ (VI). The gel concentration was 20% polyacrylamide, in 100 mM NaCl, 5 mM MgCl2 with 20 mM acetic buffer, pH 5, at 25°C. The voltage was 200 V for 3 h.

FIGURE 4

Downfield region of the 1H NMR spectra of cc+ag at 500 MHz.

FIGURE 5

Expanded regions of the NOESY spectrum of cc+ag in D₂O at pH 6.0, 35°C acquired at 600 MHz with a 240 ms mixing time. (a) A₃H8↔T₃H1′, (b) G₄H8↔C₄H1′, (c) A₅H8↔T₅H1′, (d) G₂H8↔T₃H1′, (e) A₃H8↔C₄H1′, (f) G₄H8↔T₅H1′, and (g) A₅H8↔C₆H1′.

FIGURE 6

Imino-imino proton region of the NOESY spectrum of cc+ag in H₂O at pH 6.0 acquired at 600 MHz at 20°C with a 120 ms mixing time. The detail descriptions are in the text.

FIGURE 7

Exchangeable proton region of NOESY spectrum of cc+ag in H₂O at pH 6.0 acquired at 500 MHz at 20°C with 60ms mixing time. (a) C₄H3↔A₃H8, (b1 and b2) C₄H3↔A₅NH₂, (c) C₄H3↔G₄H8, (d) C₆H3↔G₆H8, (el) T₁₀H3↔C₁₁H5, (e2 and e3) T₁₀H3↔C₁₁NH₂, (f1 and f2) T₁₀H3↔A₃NH₂, (f3) T₁₀H3↔A₃H2, (gl) T₈H3↔C₉H5, (g2 and g3) T₈H3↔C₉NH₂, (h1 and h2) T₈H3↔A₅NH₂, (h3) T₈H3↔A₅H2, (il) T₃H3↔C₄H5, (i2 and i3) T₃H3↔C₄NH₂, (j1) T₃H3↔A₃H8, (j2 and j3) T₃H3↔A₃NH₂, (kl) T₅H3↔C₆H5, (k2 and k3) T₅H3↔C₆NH₂, (l1) T₅H3↔A₅H8, (l2 and l3) T₅H3↔A₅NH₂, (m1) G₆H1↔C₇H5, (m2 and m3) G₆H1↔C₇NH₂, (n) G₆H1↔C₆H5, (o) G₄H1↔C₄NH₂, (p1 and p2) G₂H1↔C₁₁NH₂, (q) G₄H1↔A₅H2, (r1) G₄H1↔C₉H5, (r2 and r3) G₄H1↔C₉NH₂, (s) G₄H1↔C₄H5, (t) G₂H1↔A₃H2, and (u) G₆H1↔C₆NH₂.

FIGURE 8

(A) Stereo view of the superimposition of the 10 low energy structures of cc+ag. (B) Stereo view of cc+ag.

FIGURE 9

(A) Traditional TFO binds in the major groove of duplex DNA forming Hoogsteen basepairing, (B) TFOpur bends pyrimidine rich regions forming W-C pairing to one arm of pyrimidine section and Hoogsteen pairing to the other arm, and (C) H-DNA.