Cy3 nhs ester

Cy3-L, Cy3-R (Fig. 1a), Linker-L and Linker-R (Fig. 3a) DNA molecules for reconstituting nucleosomes for PIFE and FRET experiments were prepared by PCR with Cy3 labelled oligonucleotides from plasmid containing the Widom 601 nucleosome positioning sequence42 (link) with the Gal4-2C-binding site (5′-CCGGAGGGCTGCCCTCCGG-3′)65 (link) at bases 8–26 (Supplementary Table 1). Labelled oligonucleotides were prepared with Cy3 NHS ester (GE healthcare) at an amine-modified internal thymine and then HPLC purified on a 218TC C18 column (Grace/Vydac). Following PCR amplification, DNA constructs were purified by HPLC on a Gen-Pak Fax column (Waters).

A vial of Cy3-NHS ester (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was dissolved in 8 μl of dry DMSO and mixed with 500 μl of EGF (150 nmol/ml in 0.1 M sodium bicarbonate buffer, pH 8.5) in darkness for 2 h (room temperature) to synthesize Cy3-labeled EGF. Cy3-EGF was then separated from excess, unconjugated Cy3 by SEC (Sephadex G25 mini-column, pH 7.4 PBS as elution buffer), the concentration of which was calculated using the Beer-Lambert law (ɛ_Cy3 = 1.5 * 10⁵ M⁻¹·cm⁻¹). Cy3-EGF-Au NP were prepared using the same method described above using a molar mixing ratio of Cy3-EGF:Au of 160.

To identify the genes regulated by RelA/(p)ppGpp during glucose starvation, microarray analysis was performed. Overnight cultured cells were washed with CDM for three times and resuspended in CDM containing 0.2% glucose and CDM containing 1% glucose to the density of OD600 ≈ 0.1 respectively. Cells in early exponential growth period (5 h in Fig. 4) were collected for microarray analysis. The test of each strain included three biological replicates. Briefly, total RNA were isolated and purified using QIAGEN RNeasy Mini Kit (Qiagen, Shanghai, China) according to the manufacturer’s instructions. 2 μg RNA was reverse-transcribed into cDNA, and then the cDNA was transcribed into aaUTP labeled cRNA and purified. 4 μg cRNA was labeled with Cy3 NHS ester (GE healthcare) and purified. After fragmentation of the Cy3-cRNA, hybridization was performed using a Gene Expression Hybridization Kit (Agilent Technologies, Beijing, China). Finally, arrays were scanned by Agilent Microarray Scanner System with resolution of 5 μm. The signal intensities were normalized using Feature Extraction Software (Agilent Technologies) and transformed into log2 values. The microarray data have been submitted to the NCBI Gene Expression Omnibus (GEO) functional genomics data repository under the accession number GSE70092.

BsSMC stock solution was supplemented with a 1.1-fold excess of Cy3 NHS ester (GE Healthcare) dissolved in dimethylsulphoxide and left overnight at 4 °C. Free dye was removed using Micro Bio-Spin P-30 Gel Columns (Bio-Rad). We found that this step was crucial, since using a centrifugal concentrator to remove free dye abolished the protein's activity. After running the sample on an SDS–PAGE gel, the labelling was confirmed using a Typhoon imager (GE Healthcare), and the labelling efficiency was calculated by measuring absorbance at both 280 and 550 nm using a NanoDrop spectrophotometer (Thermo Scientific). The labelling efficiency was around 50% (approximately one Cy3 dye per BsSMC dimer).
The compaction rate with unlabelled WT BsSMC (Fig. 4a) was used to gauge activity of the labelled WT BsSMC. When a high concentration of Cy3-labelled BsSMC was flowed in, a spectrally distinct quantum dot attached at the end of the flow-stretched DNA was imaged onto the other half of an electron multiplying charge coupled device (EMCCD) camera. The position of the quantum dot was determined by Gaussian fitting and used to determine the compaction rate. The rate of compaction by labelled BsSMC was comparable to that by unlabelled BsSMC, indicating that labelling does not disrupt the DNA-compacting activity of the protein.

Recombinant human EGF was obtained from PeproTech (London, UK). Hydrogen tetrachloroaurate(III) hydrate (HAuCl₄) was purchased from Alfa Aesar (Heysham, Lancashire, UK). ¹¹¹InCl₃ was purchased from Mallinckrodt (the Netherlands). Cy3-NHS ester was obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Vectashield mounting medium with DAPI was purchased from Vector Laboratories (Peterborough, UK). Thermanox plastic coverslips (13 mm diameter) were purchased from Nalge Nunc International (Rochester, NY, USA). Matrigel was purchased from Corning (Tewksbury, MA, USA). MDA-MB-468 cells were purchased from ATCC. 231-H2N cells were a kind gift from Dr Robert Kerbel. All other chemicals were purchased from Sigma-Aldrich (Dorset, UK).