Adenosine-5´-mono-phosphate (AMP, Sigma, disodium salt, CAS-Nr. 61-19-8); Uridine-5´-mono-phosphate (UMP, Alfa Aesar, disodium salt, CAS-Nr. 58-97-9); Cytidine-5´-monophosphate (CMP, Alfa Aesar, disodium salt, CAS-Nr. 6757-06-8); Guanosine-5´-mono-phosphate (GMP, Alfa Aeasar, disodium salt, CAS-Nr. 85-32-5); L-Arginine (Alfa Aesar, CAS-Nr. 74-79-3); Quinacridone (Clariant GmbH, CAS-Nr. 1047-16-1); Anthraquinone (Fluka, CAS-Nr. 84-65-1); Carbon nanopowder (<50 nm (TEM); Sigma Aldrich, CAS-Nr. 7440-44-0); Carbon (mesoporous, Sigma Aldrich, CAS-Nr. 1333-86-4); Iron (II, III) oxide (Sigma Aldrich, CAS-Nr. 1317-61-9); Silicon dioxide (-325mesh, Sigma Aldrich, CAS-Nr. 60676-86-0); Polyethylene (<400 micron, Alfa Aesar, CAS-Nr.9002-88-4).
3ml aqueous suspensions were made of each 0.1 g/ml inorganic substrate (e.g. graphite powder) and/or organic pigment with a total nucleoside monophosphate concentration of 50mM. Samples were incubated over night at 60°C while mixed horizontally at 300 rpm, to avoid sedimentation of substrates and pigment. After incubation, samples were centrifuged at 8000x g, 1 min, at room temperature (RT). The supernatant was transferred to a new collection tube.
Precipitation was carried out with 0.2 M NaCl and 3.5 volumes of EtOH for isolation of short RNAs (2–10 nt), and with 0.3 M NaOAc (pH 5.2) and 0.7 volumes of isopropanol for isolation of longer RNAs (> 10 nt). After addition of the regarding amount of salt-solution and alcohol, samples were mixed by inverting the tubes 5 times and precipitation reactions were incubated over night at -20°C. Afterwards the samples were subsequently centrifuged at 14.000x g, 1h, at room temperature. Supernatant was discarded, leaving about 20µl of it inside the reaction tube, in addition to any formed gel pellet. Formed gel pellets were dried at 37°C for 20min and resuspended with an appropriate amount of H2O dest.
Concentrations of miRNA suspensions were measured using a Qubit® 3 Fluorometer (Invitrogen™), and the Qubit® microRNA Assay Kit (Invitrogen™) or the Qubit® RNA HS Assay Kit (Invitrogen™)36 due to its high specificity and reliability37. Concentrations were calculated by the chosen miRNA or RNA program of the Qubit® 3 Fluorometer. Standard curves and samples were prepared, following the manuals38. Concentrations were calculated and normalized regarding the respective volume of each sample.
For RT-qPCR selected samples were reverse transcribed using the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher) following the manual. As a positive control the synthetic miRNA hsa-miR-134-3p (5´-phosphorylated) (eurofins) with an oligonucleotide length of 23 nt was used. As a negative control nuclease-free water was used. For qPCR the Master Mix reaction was composed of 10µl QuantiTect SYBR® Green (Qiagen), 2µl amplification primer mix from the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher), and 8µl of diluted sample. qPCR was ran on a LightCycler® 480 Instrument II with the following settings: 15min 95°C, 40x: 15s 94°C – 30s 60°C – 30s 72°C – Single Data Acquisition, 72°C 2 min, Melting curve.
Scanning tunnelling microscopy
For Scanning Tunnelling Microscopy (STM) investigations aqueous suspensions were prepared of each 2% w/w of quinacridone pigment and a nucleotide monophosphate or amino acid final total concentration of 25mM, if required. Suspensions were drop cast to graphite substrates to induce OSWD16 and dried overnight. The samples were subsequently covered with dodecane to achieve a defined, stable environment between STM tip and substrate. Each sample was prepared twice. From each replicate 15 images of 200nm x 200nm size were generated from 3 different spots. Images were acquired with a self-build STM combined with a SPM 100 control system, supplied by RHK Technology. The scans settings were: bias = 1 V, tunnel current 300 = pA, line time = 50 ms. Further, the voltage pulses used to improve the scan quality were applied within the range of 4.3 V to 10 V. All STM measurements were performed under ambient conditions.
Surface coverage determination
To determine coverage, meaning how much area of graphite is covered by supramolecular QAC adsorbate structures (Extended Data Fig. 1), black/white histograms were created. For this purpose, depicted QAC adsorbate structures were masked out in the image (white) while setting the background (graphite without QAC adsorbate structures) to black (Extended Data Fig. 2). Black/white histogram values were calculated using “histogram” within the “analyse” menu of the software ImageJ. The mean values given in the histogram data box were then converted to percentages of the graphite area covered with QAC adsorbate structures, whereby a histogram value of 0 means 0% of the graphite surface is covered with adsorbate and a histogram value of 255 means 100% of the graphite surface is covered. Note that AMP itself does not form monolayer on graphite due to its 3D-structure. For statistical analysis of the coverage results t-test with Welch's corrections was used since the samples show unequal distribution variance. The calculations were performed using Excel, giving the results of two sided unequal variances t-test.
All molecular mechanics calculations were performed with the Materials Studio package (Accelrys). The force field used was Dreiding39 where partial charges of atoms within the molecule are calculated with the Gasteiger method40. Geometry optimization calculations were calculated where convergence tolerance regarding to energy was 1.0e-4kcal/mol, to force was 0.005kcal/mol/Å and to displacement was 5.0e-5 Å. The Smart algorithm was used which is a concatenation of steepest descent, Newton-Raphson and quasi-Newton methods to get a better behaviour for the different stages of downstream minimization. Dynamics calculations were performed for a NVE ensemble, with a temperature of 298 K, with random values assigned for the initial velocities of the atoms and with a time step of 0.1fs for the integration algorithm.
A chain of 12 AMP molecules was constructed starting with building an AMP dimer. The dimer itself was built by duplicating an AMP and performing an 180° rotation related to the axis lying in the plane of the adenine molecule and passing through the centres of the hexagon and pentagon. Afterwards, the rotated AMP was translated normal to the plane of the adenine by a distance of 3.5 Å. This dimer was stacked 6 times in a row such that all planes of the adenine molecules were parallel each by a distance of 3.5 Å. This artificially constructed model was finally optimized related to geometry.
The entire system is modelled within a supercell containing two graphene layers, on top in some distance a chain of AMP molecules and finally two layers of an QAC crystal above the AMP chain. The entire system was filled with water molecules for different concentrations. The initial position of the AMP chain related to the graphene layers and the QAC crystal layers is stated below for the different calculations performed. The vertical dimension of the supercell has been chosen to be safe that an interaction of the graphene layers and the QAC crystal layers can be neglected (gap is 43 Å). The rectangular in-plane dimensions of the supercell have been determined to minimize the distortion of the periodically structure of the QAC crystal layers. The deviation of the unit cell vectors of the QAC layers are almost zero in one direction (0.1%) but quite large in the other direction (5.6%). However, because the outer layer of the QAC crystal is always kept fixed, the adjacent free movable QAC layer was observed stable throughout all simulations. This observation was the reason why this significant deviation was accepted for all calculations. The graphene layers are not distorted with regard to the optimal unit cell dimensions. Dimensions of the supercell are: plane x, y: 80.956 Å, 56.58 Å, z: 103.4 Å.
36. Determine microRNA concentration in solution: Fluorescence-based small RNA quantitation for both conventional and high-throughput assays, BioProbes® 70, 32-36 (2014).
37. Garcia-Elias, A., Alloza, L., Puigdecanet, E., Nonell, L., Tajes, M., Curado, J., Enjuanes, C., Díaz, O., Bruguera, J., Martí-Almor, J., Comín-Colet, J., Benito, B. Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs. Rep. 7(1), 1-14 (2017).
38. Thermo Fisher Scientific Inc., “Qubit® 3.0 Fluorometer”. Publication MAN0010866. http://tools.thermofisher.com/content/sfs/manuals/qubit_3_fluorometer_man.pdf (2014).
39. Mayo, S. L., Olafson, B. D., Goddard, W. A. DREIDING: a generic force field for molecular simulations. Phys. Chem. 94(26), 8897-8909 (1990).
40. Gasteiger, J., Marsili, M. Iterative partial equalization of orbital electronegativity – a rapid access to atomic charges. Tetrahedron 36(22), 3219-3228 (1980).