Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

Abstract

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K⁺ in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na⁺, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

Keywords: G-quadruplex; fluorescence resonance energy transfer; optical tweezers; single-molecule biophysics.

Fig. 1.

Conformational analysis of human telomeric repeats (hTel22). (A) CD spectrum in 100 mM K⁺. (B) Schematic diagram of hTel22 construct for smFRET. The 5′ extension to the 22-nt-long human telomeric repeat GGG(TTAGGG)₃T was annealed to a 18-nt-long biotinylated strand and immobilized on a PEG-passivated quartz surface through biotin–neutravidin interaction. The 5′ end of the biotinylated strand is labeled with Cy5 (acceptor). The main G4 strand is labeled with Cy3 (donor) at the 3′ end of the telomeric repeat and is followed by (dT)₁₇ and an 18-nt extension that is annealed to a 30-nt-long λ-bridge. (C) E histograms as a function of K⁺ concentration. (D) Representative single-molecule time traces of donor and acceptor intensities and corresponding E in 100 mM K⁺ (30-ms integration time).

Fig. 2.

Conformational dynamics of hTel22 under tension in 100 mM K⁺. (A) The G4 strand is annealed to a biotinylated strand and immobilized on a neutravidin-coated quartz surface. The other end is connected to a 1-μm-diameter, optically trapped bead through a λ-DNA. The G4 construct was stretched to ∼28 pN in 6.5 s. FRET was measured between Cy3 (donor) and Cy5 (acceptor) as a function of force. (Inset) Unfolding (black) and refolding (red). (B) A representative single-molecule time trace (20-ms integration time) of donor and acceptor intensities and corresponding E. Stretching and relaxation occurred at a stage speed of 455 nm/s. (C) f_unfold and f_refold in different stretching cycles from a single molecule shown in B. (D and E) Distributions of f_unfold (D) and f_refold (E). The red and black curves in D, respectively, denote individual and overall rupture force distributions predicted using the Dudko–Szabo model (44, 45). Only those cycles showing complete unfolding were included (n = 188). (F) E vs. force example curves, one representative from each of the four peaks in f_unfold distribution (i–iv), one representative of a ultrastable state (v), and one representative of gradual partial unfolding/folding without hysteresis (vi). (G and H) E vs. force curves of (dT)₂₂ (G) and hTel22 midE population (H). Corresponding E histograms are shown in insets. Error bars represent SEs.

Fig. 3.

Mechanical unfolding of hTel22 in 100 mM K⁺. (A) Dual optical trap experimental scheme. The G4 strand is sandwiched between two 1.5- and 1.7-kb-long dsDNA spacers to the left and right, respectively. A (dT₁₇) sequence is inserted between the spacers and the GQ-forming sequence to minimize any influence of base stacking on mechanical unfolding. The spacers are linked to beads (diameter ∼1 μm) via digoxigenin–anti-digoxigenin and biotin–streptavidin linkages, respectively. The G4 construct was stretched to ∼30 pN in 3.5 s. (B) Representative force vs. extension response from a single molecule in four consecutive stretching/relaxation cycles (100 mM K⁺). Black and red curves represent GQ unfolding and refolding, respectively. The folded and unfolded WLC fits are respectively denoted by black and red dashed lines. Black and red arrows indicate unfolding and refolding events, respectively. (C) f_unfold vs. cycle number for the molecule shown in B. (D) f_unfold values obtained from 345 cycles from 71 molecules are widely distributed within each molecule. Low force unfolding below the detection limit of ∼3 pN, marked with black dashed line, is shown as a gray circle at 0 pN. The red dashed lines indicate the force limit (∼28 pN) achievable with the fluorescence-force spectroscopy assay. (E and F) Distributions of f_unfold (D) and f_refold (E) for 345 cycles from 71 molecules. Only those cycles showing complete unfolding were included (n = 277). The red and black curves, respectively, denote individual and overall the rupture force distributions predicted using the Dudko–Szabo model (44, 45). (G) f_unfold across consecutive pulling cycles shows weak or no correlation. (H) Coefficient of variation for f_unfold calculated for the entire data set and within individual molecules (averaged over all molecules).

Fig. 4.

Extreme diversity of hTel22 in Na⁺. hTel22 behavior in 100 mM (A–D) and 10 mM Na⁺ (E–G). (A) E histogram in 100 mM Na⁺ in the absence of force. (B) A representative E time trace during two stretching/relaxation cycles. (C and D) Distributions of f_unfold and f_refold (n = 197 for both). The red and black curves in C, respectively, represent individual and overall rupture force distributions predicted using the Dudko–Szabo model (44, 45). (E) E histogram of GQs in 10 mM Na⁺ in the absence of force. (F) A representative E time trace during four stretching/relaxation cycles. (G) E vs. force example curves. B and F were acquired at 20-ms integration time. Error bars represent SEs.

Fig. 5.

Single-point mutant of human telomeric repeats (hTel22_mut) exhibits GQ-like behavior in 1 M K⁺. (A) E histograms hTel22_mut as a function of K⁺ concentration in the absence of force. Red arrow indicates initiation of secondary structure formation at 150 mM K⁺. (B and C) Representative E time traces (20-ms integration time) during stretching/relaxation in 100 mM (B) and 1 M K⁺ (C). (D and E) Distributions of f_unfold and f_refold of molecules showing abrupt unfolding transitions (n = 56). The black and red curves, respectively, denote an overall and individual rupture force distributions predicted using the Dudko–Szabo model (44, 45). Data presented in C–E were collected in 1 M K⁺.

Fig. 6.

G-triplexes behave like ssDNA. (A) E histograms of hTel16 as a function of K⁺ concentration. (B) A representative E time trace in 100 mM K⁺ (30 ms integration time). Average E vs. force curves of hTel16 (C) and (dT)₁₆ (D) in 100 mM K⁺. Error bars represent SEs.

Fig. 7.

Values of (A) Δx^‡, (B) τ_u(0), and (C) ΔG^‡s estimated for the f_unfold clusters observed from fluorescence-force spectroscopy data of hTel22 in 100 mM K⁺.