No labelling was required as the PNA-library that was screened with the MRSA and MSSA cells was a synthetic combinatorial PNA library, which was synthesized with a fluorescent tag: 5(6)-carboxyfluorescein (FAM). This tag was covalently linked to the PNA and the FAM-PNA constructs stay intact during cellular incubation. This allowed extraction of the intensity of the FAM label, thereby giving the relative amount of PNA hybridized to each spot on a complementary DNA microarray to be quantified. (Parameters: Amplification = none, Mass unit = Micro gram)
DNA arrays (Oxford Gene Technologies, OGT) complementary to the Library-1 were hybridized with the PNA-conjugates purified from cells in 1 x GenHyb buffer (110 uL, Genetix) buffer overnight from 50C to 32C. As a control untreated 1296-peptide/PNA (10 uM) library was hybridized onto a complementary DNA-microarray in the same manner. The arrays were washed with Oxford Gene Buffer (100 mM NaCl, 40 mM citric acid, 0.7% (w/v) N-lauroylsarcosine sodium salt, 0.1 mM EGTD, pH 8.5) for 10 min at 30C, rinsed with 3 x H2O, Tris buffer (10 mM, pH 8.5), and dried under N2 flow. Microarrays were scanned with a Tecan LS Reloaded microarray scanner with ArrayPro 32 Analyzer (Media Cybernetics Inc.) software and a FITC filter. Analysis of the microarray images was performed using Bluefuse software. (Parameters: Chamber type = OTHER: Agilent Hybridization Chamber, Quantity of label target used = 5, Mass unit = Micro gram, time = 24, Tiny time unit = hours, Volume = 110, Volume unit = Micro litre, temperature = 32)
Microarrays were scanned with a Tecan LS Reloaded microarray scanner with ArrayPro 32 Analyzer (Media Cybernetics Inc.) software and a FITC filter. Analysis of the microarray images was performed using Bluefuse software. (Parameters: Scanning hardware = OTHER: Tecan LS Reloaded, Scanning software = Scanning software)
Raw microarray data was obtained from Bluefuse, which allows grid alignment and signal estimation, in an Excel format. In excel the top ~5% and the bottom ~5% of each of the 33 replicate-sets were removed as outliers (erroneous values caused by dust, scrapes etc). The mean fluorescence intensity was calculated over each of the remaining 29 replicates and over the non-complementary DNA features (negative controls). Identification of generic consensus delivery agent: The 1296 mean intensities were corrected for the microarray background by subtracting the mean intensity of the non-complementary negative control features. Hereafter, the data was normalized for the control hybridization by calculation the ratio between the background corrected mean intensities of the data sets obtained from hybridization of PNA extracted from cells and the data set obtained from the hybridization of the untreated 1296-peptide/PNA library. Clustering analysis: To allow comparison of the data sets from different arrays these were normalized so that the data sets had the same mean intensity arbitrarily set to 100 and the 1296 mean intensities in each dataset were multiplied with respective normalization factors. Hereafter, the normalized intensities for the 5 cell types [MRSA, MSSA, Lymphocytes, Monocytes, and Neutrophils] were clustered using hierarchical clustering based on a Euclidean metric using the "scipy.cluster" Python package. The resulting dendrogram was cut on a user-defined basis such that 20-30 clusters were produced. Single cell type based clustering analysis: The 1296 normalized intensities were separated into 5 groups; one group for each cell type such that all the peptides that lead to maximum for one specific cell line were compiled in one group. The clustering analysis was performed on each of the 5 datasets as described above leading to a dendrogram and a heat diagram for each cell type.
MRSA (1.6 x 10^9) and MSSA (1.9 x 10^9) cells were washed with PBS (x 3) and incubated with Library-1 (50 uM) in PBS supplemented with 1% BSA (1 mL) under gentle shaking at 37C for 30 min. (Parameters: start time = 30, time unit = minutes, min temperature = 37, temperature unit = C)
After incubation MRSA and MSSA cells were collected by centrifugation (13000 rpm for 1 min), washed with PBS supplemented with 1% BSA and 1% Tween 20 (x 3), and lysed with 1% Tween 20 and 1% SDS in H2O (1 mL) under gentle shaking at 99C for 10 min. The extracted peptide-PNA conjugates were purified by filter-centrifugation separating between 3,000 Da and 10,000 Da molecular weight filters (Nunc TM) and concentrated by speed-vac. The amount of extracted PNA was estimated using a NanoVue spectrometer: MRSA (4.72 ug) and MRSA (2.77 ug). (Parameters: Extracted product = OTHER: peptide nucleic acid, Amplification = none)