Silanization and biotinylation
Glass slides (Menzel Gläser, Menzel GmbH & Co. KG, 38116 Braunschweig, Germany) were cleaned with ultrasound in acetone for 15 minutes and again in ethanol (acetone and ethanol were obtained from Carl Roth GmbH & Co. KG, Karlsruhe, Germany). After rinsing with ultrapure water, the slides were put into NaOH (10 M) for 1 minute and washed thoroughly with water. Drying was carried out in a centrifuge (Varifuge 3.0R, Heraeus) for 1 minute. In the vapor phase at 120°C silanization with 3-Aminopropyltriethoxysilane (Fluka Chemie GmbH, 89552 Steinheim, Germany) was executed in a sealed beaker and finished after 60 minutes. For biotinylation, Sulfo-NHS-Biotin (20 mg) (Thermo Scientific, IL 61101 USA) was dissolved in DMSO (1 mL) (Carl Roth GmbH & Co. KG) because of its low stability and moisture-sensitivity. The DMSO solved Sulfo-NHS-Biotin can be stored at -20°C with desiccant.
Sulfo-NHS-Biotin (10 mL) solution was added to Na2HPO4 (100 mM, 21 mL), NaCl (150 mM) buffer at pH 7.4. Incubation of 5 silanized glass slides took place for 3 hours at room temperature. Slides were washed with PBS and rinsed with water. Blocking was carried out by incubating the glass slides in a freshly prepared, 0.1% (w/v) solution of blocking reagent CA from Applichem in 100 mM Tris-Cl. For cleaning, slides were washed three times for 5 minutes in Tris-Cl (100 mM Tris, 600 mM NaCl, pH 7.4) and finally rinsed with ultrapure water. NaOH, Na2HPO4, NaCl, PBS and blocking reagent CA were obtained from AppliChem GmbH, 64291 Dortmund, Germany.
DNA-Array preparation
Neutravidin (Thermo Scientific, IL 61101 USA) that had been spotted had to be addressed by biotinylated oligonucleotides (Biomers.net GmbH, 89077 Ulm, Germany). Sequences of the oligonucleotides: Side A LcF5: 5'-CTT ATC GCT TTA TGA CCG GAC C-3' (5': Biotin); Side B RcF6: 5'-CAA TGA AAC ACT AGG CGA GGA C-3' (5': Biotin). Staining of the outer frame was done with biotinylated DY-547 dye (Dyomics GmbH, 67745 Jena, Germany). All these three components were diluted in carbonate buffer pH 9.0 to a final concentration of 1 mM. Incubation time for binding was 5 minutes and was stopped by washing with 1× PBS-buffer and ultrapure water. The left DNA strand M13-L part and right DNA M13-R strand were diluted 1:50 and 5 μl of each solution were transferred onto the chip directly to the prepared array. After incubation in the dark for 60 minutes in TE-buffer at 37°C and 85% rel. humidity, the glass chip was washed by completely dipping it into PBS-Tween and rinsing it a second time in PBS.
Spotting
An atomic force microscope CP-II from Veeco (Santa Barbara CA, 93117 USA) and AFM-tips from NanoSensors (NanoAndMore GmbH, 35578 Wetzlar, Germany) were used: DT-CONTR (force constant: 0.2 N/m; resonance frequency: 13 kHz). Movement of the AFM-tip and execution were controlled by the diNanolithography Software V.1.8. Approaching the biotinylated glass slide was achieved in contact mode with 3.4 mN contact force. The tip remained in contact for 4 seconds and changed to the next spotting positions by retraction. Ink was supplied to the tip by a hypodermic needle of Popper & Sons, Inc. (N.Y. 11040 USA).
DNA preparation
The DNA-construct was generated by digesting 10 μg M13mp18 RF I DNA plasmid (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) simultaneously with the restriction enzymes PstI, Acc65I and BamHI (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) in NEBuffer-3 at 37°C for 2 h. Then the enzymes were inactivated by heating the batch to 80°C for 20 minutes and finally cooling down slowly (1 K/min.). Parallel to this, hybridization of the adapter segments in Tris-Cl buffer (100 mM Tris-Cl; 600 mM NaCl; pH 7.4) took place by heating the oligonucleotides up to 90°C for 5 minutes (see figure 4) and cooling down slowly (1 K/min.). The digested M13mp18 plasmid (120 μl) was then divided into a left and right batch. The left was incubated with 8 μl M13-L5 (10 μM) and 8 μl M13-M2 (10 μM). The right was incubated with 8 μl M13-R6 (10 μM) and 8 μl M13-M1 (10 μM). Prehybridization took place for 30 minutes at 40°C, then 30 minutes at 30°C followed by cooling down to 20°C. Both batches were then ligated separately with T4 DNA ligase (New England - BioLabs GmbH, 65926 Frankfurt a. M., Germany) over night at 4°C. To avoid rupture of the sensitive construct, ligation was not stopped by heating but by removing the enzyme by cleaning it with Sure Clean (Bioline GmbH, 14943 Luckenwalde, Germany) and dissolving it in 100 μl TE-buffer (50 mM Tris-Cl, 100 mM NaCl). The concentration of both, the left and the right batch, were equalized by adding TE-buffer to a final concentration of about 30 ng/μl. The product was then stored at -20°C.
PNA synthesis
The PNA 3927 was synthesized by conventional solid phase Boc chemistry as previously described [24, 25], and purified by reversed phase HPLC. The PNA was subsequently characterized by HPLC and MALDI-TOF mass spectrometry (see additional file 2). Furthermore, the thermal stability (T
m
) of complexes with an oligonucleotide (5'-GAG GGA AGG-3') binding to the triplex domain and an ologonucleotide (5'-CAT CCA CAG GGG TAA-3') was determined as 87°C and 77°C, respectively (see additional file 3), showing that both domains are functional in terms of hybridization to a DNA target.
Microarray test
Glass slides (Menzel Gläser, Menzel GmbH & Co. KG, 38116 Braunschweig, Germany) were blocked 1 h with 0.1% blocking reagent CA (AppliChem GmbH, 06466 Gatersleben, Germany) after they were silanized and biotinylated as described above. The reactive glass slides were incubated over night with 25 ng/ml Avidin at room temperature. Microarrays were spotted contactless with the microarray spotter TopSpot (BioFluidiX GmbH, 79110 Freiburg, Germany) on the functionalized and blocked glass slides. The solutions that have been spotted were: 2.7 μl left DNA construct, 2.7 μl right DNA construct, 2.7 μl PNA-adapter and either 2.7 μl LcF5-Btn 1 μm or 2.7 μl RcF6-Btn 1 μm. For a negative sample one component was omitted (PNA or adapter oligonucleotide). Incubation took place at 25°C at 85% rel. humidity for 1 h. For detecting the PNA and the DNA's orientation a Cy5 labelled oligonucleotide (Cy5-cF6; 1 μl) and a Cy3 labeled oligonucleotide (Cy3-cF5; 1 μl) were hybridized at 35°C at 85% rel. humidity for 1 h and were finally detected by a fluorescence microarray scanner (Axon Instruments, GenePix 4200A).
Microscopy
Fluorescence microscopy was carried out with an upright epifluorescence microscope Olympus A BX51 (objective: UPlanFL N; 40 × 0.75). Fluorescence detection was accomplished with the following filter-cube combinations: DY-547 detection: excitation filter (Ex) BP 545/25, dichromatic mirror (Dm) 565, emission filter (Em) LP 605/70 and for SYBR-Green I detection: Ex BP 460 - 495, Dm 505, Em LP 510 - 550. For illumination a mercury arc lamp (100 W, OSRAM GmbH, 81543 München, Germany) in combination with a Uniblitz VCM-D1 shutter was used. Image acquisition was carried out with a CCD camera (FView II) with 12 bit dynamic range and 1376 × 1032 pixel resolution. Software aquisition was donw with cellˆR version 3.1 (build 1276). Image editing was realized with ImageJ V1.42q. Staining of DNA was performed with SYBR-Green I (1:10000 in DMSO).