Construction of plasmids

A pair protein for FRET, RHAU55-CFP/RHAU55-YFP, was generated by fusing the RHAU peptide (55 aa) with CFP and YFP. DNA encoding the RHAU peptide was amplified by PCR using the gene as the template and the primer pair ON5/ON6 (ON5: 5'- gcg tgg atc cgt cca tgc atc ccg ggc acc tga aag-3"; ON6: 5'- gat tca tat ggc tgc cgc cgc cgc tct tcg ctt gaa cag aat tca gta ac-3'). The PCR product was inserted into treated pETDuet1 (Merck Millipore, Germany) at the BamHI and NdeI sites, resulting in the generation of the plasmid pRHAU. DNAs encoding YFP and CFP were amplified by PCR using pHT58430 containing the gene and pHT58240 containing the gene as templates and the primer pair ON7/ON8 (ON7: 5’-tta aag atc tag gcg gcg gca gca tgg tga gca agg gcg agg ag-3’; ON8: 5’-cca tct cga gtt act tgt aca gctcgt cca tgc cga gag tg-3’). The PCR products were then inserted into treated pRHAU at BglII and XhoI sites, resulting in the generation of plasmids pRHAU55-YFP and pRHAU55-CFP, respectively.

A plasmid for the expression of the negative control protein CFP was genetically generated by directly cloning the gene into the vector pETDuet1. DNA encoding the CFP protein was amplified by PCR using the gene as the template and the primer pair ON9/ON10 (ON9: 5'- tta agg atc caa tgg tga gca agg gcg agg ag-3'; ON10: 5'- tgc cat atg gtt act tgt aca gct cgt cca tgc c -3'. This PCR product was inserted into treated pETDuet1 at the BamHI and NdeI sites, resulting in the generation of the plasmid pCFP.

Expression and purification of proteins:

The plasmids pRHAU55-YFP (coding RHAU55-YFP), pRHAU55-CFP (coding RHAU55-CFP) and pCFP (coding CFP alone) were chemically transformed into BL21(DE3). The were cultured in Luria-Bertani medium (10 g NaCl, 5 g yeast extract and 10 g peptone in 1 L Milli-Q water) with 100 mg/L ampicillin, and the cells were incubated at 37 °C with shaking at 220 rpm to reach an OD600 of 0.8-1, IPTG (0.3 mM) (Sigma Aldrich, St. Louis, MO, USA) was subsequently added to the cell culture. The cells were continuously incubated for 12 hours at 16 °C and shaken at 180 rpm before being harvested. The pellet of cells was then resuspended in a reagent (bugBuster protein extraction, EMD Millipore, Burlington, MA, USA) consisting of bezonase nuclease (DNA and RNA degradation). The insoluble material was removed by centrifugation at 18,000 rpm for 1 hour at 4 °C. The soluble supernatant was added to the His-tag column (ThermoFisher Scientific, Waltham, MA, USA). After that, the His column was washed with 30 volumes of the column with washing buffer (20 mM Tris-HCl, 100 mM NaCl and 10 mM imidazole, pH 7). The target proteins were finally eluted with lysis buffer (20 mM Tris-HCl, 100 mM NaCl and 200 mM imidazole). The imidazole chemical in the protein solution was then removed by centrifugation using the Amicon Ultra15 (EMD Millipore). The pure target proteins were collected and analyzed by SDS–PAGE.

Circular dichroism spectroscopy

Circular dichroism (CD) measurements for the determination of the TERRA structure were performed on a spectropolarimeter (JASCO-815) using a path length quartz cuvette (10 mm) in a 40 µl reaction volume at 25 °C. Scanning from 220 nm to 320 nm was performed at a rate of 200 nm/min, pitch (1 nm), and bandwidth (2 nm). The TERRA sample was measured at concentrations of 10 µM in a buffer of 20 mM sodium phosphate and 100 mM KCl, pH 7. An average of two scans were taken for each measurement.

FRET measurements

All samples for FRET were measured in a buffer solution consisting of 20 mM potassium phosphate, pH 6.5, in quartz cuvettes with a 10 mm path length (Hellma). The sample of proteins was used at a concentration of 1 µM for the measurement. All of the FRET measurements were performed using a Cary Eclipse spectrophotometer (Varian), and all fluorescence data were automatically recorded at room temperature with a 410 nm excitation wavelength. All parameters of measurements were kept constant over the measurements to ensure all data comparison.

Construction of plasmids, protein expression and purification

The RHAU peptide bearing 16 amino acids (aa 53-68) is able to recognize and bind a parallel G4; however, the RHAU peptide length also influences G4 binding affinity . In this study, the fluorescent protein FRET RHAU55-YFP/RHAU55-CFP pair was generated by fusing 55 amino acids of RHAU peptide (53-107) to YFP/CFP. All DNA sequences encoding RHAU55-YFP, RHAU55-CFP and CFP were analyzed and confirmed by DNA sequencing. Proteins were expressed in the BL21 (DE3) upon the addition of IPTG. Proteins bearing a His-tag at the N-terminus were purified by chromatography using a His-tag column. The electrophoresis of proteins on SDS–PAGE showed that pure target proteins were collected (Figure 2). The molecular weights of RHAU55-YFP and RHAU55-CFP are 36,3 Da and 36,2 Da, respectively, which relocated between the bands of 30 kDa and 40 kDa of the ladder. The reference CFP is 28,6 Da, which relocated between the bands of 25 kDa and 30 kDa of the ladder.

Circular dichroism spectroscopy

CD spectroscopy as a primary method was applied to characterize the G4 structure to evaluate the TERRA 9 repeat structure topology. TERRA RNA UU(AGGGUUA) was treated with 100 mM KCl for CD measurement. The CD spectrum of TERRA, measured at a concentration of 10 µM, displayed a positive peak at 265 nm along with a trough at 245 nm (Figure 3). This confirmed that TERRA 9 repeats formed a parallel G4 topology under K conditions.

Fluorescence resonance energy transfer (FRET) measurements

Herein, we used the hetero-FRET system to evaluate the binding mode of TERRA 9 repeats G4 and RHAU peptides. Incorporating RHAU peptide into a donor CFP and an acceptor YFP resulted in the construction of a pair of fluorescent protein FRET RHAU55-CFP/RHAU55-YFP, respectively. The addition of TERRA 9 repeats G4 (1 µM) to a mixture of RHAU55-CFP (1 µM) and RHAU55-YFP (1 µM) resulted in the occurrence of a strong hetero-FRET (Figure 4a). In fact, the hetero-FRET signal is obtained from 50% heterodimerization, while the remaining 50% homodimerization of protein does not donate to the hetero-FRET signal in the mixture of TERRA 9 repeats and proteins. The addition of TERRA 9 repeats G4 (1 µM) to a mixture of RHAU55-CFP (1 µM)/RHAU55-YFP (1 µM) led to an increase in the peak ratio of 527 nm/475 nm from 0.52 to 1.72 (Figure 4b). Hetero-FRET was physically observed in TERRA 9 repeat G4-dependent concentrations. The FRET signal increased with increasing concentrations of TERRA 9 repeats G4 (Figure 4a). These results showed that TERRA 9 repeats G4 could bind two RHAU peptides simultaneously. In contrast, the addition of TERRA 9 repeats G4 (1 µM) to a solution of reference CFP (1 µM) and RHAU55-YFP (1 µM) did not increase the peak ratio 527 nm/475 nm (Figure 4b). HeteroFRET was not observed in the mixture of CFP/RHAU55-YFP in the addition of TERRA, which revealed that TERRA 9 repeats G4 did not bind any side chain of CFP and YFP.