Mechanical unfolding of long human telomeric RNA (TERRA)

Abstract

We report the first single molecule investigation of TERRA molecules.By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.Telomeres are nucleoprotein structures that protect chromosome ends from being recognized as DNA breaks.1 Mammalian telomere DNA consists of long tandem arrays of double-stranded TTAGGG repeats that are maintained by the telomerase. 2 The 3 0 end of each telomere terminates in a G-rich single-stranded overhang (150-200 nucleotides) that is able to self-fold into G-quadruplexes. The demonstration of the in vivo presence of DNA G-quadruplexes at telomeres suggests that these non-canonical secondary structures have a role in telomere end-protection, and therefore in chromosome stability. 3,4 It has also been found that telomere end-binding proteins control the folding and unfolding of DNA G-quadruplexes in vitro 5 and in vivo. 4Telomeres are transcribed from the subtelomeric regions towards chromosome ends into telomeric repeat-containing RNA (TERRA). 6,7 These non-coding RNA molecules contain subtelomere-derived sequences and an average of 34 GGGUUA repeats at their 3 0 end.8 TERRA acts as a scaffold for the assembly of telomeric proteins involved in telomere maintenance and telomeric heterochromatin formation. 9,10 Importantly, there is also evidence for the presence of TERRA RNA G-quadruplexes in living cells. Here, we report the synthesis and purification of TERRA molecules up to 96nt (twice the largest molecule previously analyzed by NMR), a size similar to the endogenous nuclear TERRA 8 (see ESI †).We have also used optical-tweezers (OT) to study RNA molecules with different numbers of possible quadruplexes ranging from 1 (Q1) to 6 (Q6). CD spectra of the RNA sequence Q4 in 25 mM potassium phosphate pH 7 buffer showed a positive band at 260 nm and a negative band at 240 nm (Fig. 1a), the characteristic signature of parallel-stranded G-quadruplex conformations. This result is in agreement with previously reported observations showing that other telomeric RNA sequences are folded into parallel G-quadruplex structures both in sodium and potassium solutions. 12,13,[16][17][18][19][20] It is also important to mention that no band at 300 nm was observed for this long telomeric RNA as it was reported for 22-mer and 45-mer telomeric RNA in ammonium acetate buffer. 15 On the other hand, the normalized CD spectra of the bimolecular G-quadruplex formed by r(UAGGGUUAGGGU) and Q4 showed a similar shape and amplitude, which indicates that most of the Q4 molecules form four G-quadruplexes (Fig. 1b). 21Thermal stability of Q4 was also measured by recording CD melting curves. To test the influence of the potassium concentration on the thermal stability we performed the experiments in two different potassium-containing buffers (Fig. 1c). Melting temperatures of 65.5 1C and 72.5 1C were obtained in 2...

Mechanochemical Properties of Individual Human Telomeric RNA (TERRA) G‐Quadruplexes

“…It is interesting that the partially folded structure has a rupture force comparable to that of RNA GQ (Figure 3), which suggests that the partially folded structure is more rigid but also more fragile than the fully folded GQ. During the submission of this paper, it was reported that rupture force for a TERRA construct that contains five telomeric repeats is lower than what we have observed here 32. It is possible that the additional telomeric repeat may reduce the mechanical stability of the RNA quadruplex.…”

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

“…Optical tweezers represent a unique approach to investigate the mechanical stability of folded nucleic acid structures at the single-molecule level1438394041. This technique has been used to demonstrate the formation of G-triplex structures formed by human telomeric sequences containing three GGG repeats under dilute conditions and in the presence of Na + ion14.…”

Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G‐Quadruplex

“…The rupture-force histogram for the TERRA GQ without ligands shows more than one population (Figure 4 A ), representing multiple conformations in the same TERRA sequence as reported previously. [ 24 , 28 ] When the TERRA construct was incubated with 5 μ m cPDS, we observed an increase in the rupture forces (from 23/36 pN to 25/40 pN), thus suggesting that the binding of cPDS increases the mechanical stability of the TERRA GQ (Figure 4 B ). A similar increment in mechanical stability was observed for TERRA-G4 with 50 n m BG4 (Figure 4 C ).…”