Adenosine-5´-monophosphate (AMP, Sigma, disodium salt, CAS-Nr. 61-19-8); Uridine-5´-monophosphate (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´-monophosphate (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).
Three millilitre of aqueous suspension was made of each 0.1 g/ml inorganic substrate (e.g., graphite powder) and/or organic pigment with a total nucleoside monophosphate concentration of 50 mM. Samples were incubated overnight at 60°C while mixed horizontally at 300 rpm, to avoid sedimentation of substrates and pigment. After incubation, samples were centrifuged at 8000x g for 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 the addition of the appropriate amount of salt solution and alcohol, samples were mixed by inverting the tubes 5 times, and precipitation reactions were incubated overnight at -20°C. Afterwards the samples were subsequently centrifuged at 14.000x g, for 1 hour at room temperature. The supernatant was discarded, leaving approximately 20 µl of it inside the reaction tube, in addition to any formed gel pellet. Formed gel pellets were dried at 37°C for 20 min and resuspended in an appropriate amount of H2O dest.
Concentrations of miRNA suspensions were measured using a Qubit® 3 Fluorometer (Invitrogen™), 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 10 µl QuantiTect SYBR® Green (Qiagen), 2 µl amplification primer mix from the TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermo Fisher), and 8 µl 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 quinacridone pigment and a nucleotide monophosphate or amino acid final total concentration of 25 mM, if required. Suspensions were drop cast onto graphite substrates to induce OSWD16 and dried overnight. The samples were subsequently covered with dodecane to achieve a defined, stable environment between the STM tip and substrate. Each sample was prepared twice. From each replicate, 15 images of 200 nm x 200 nm size were generated from 3 different spots. Images were acquired with a self-built STM combined with an SPM 100 control system supplied by RHK Technology. The scan settings were: bias = 1 V, tunnel current = 300 pA, and 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 (Supplementary 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 (Supplementary 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 monolayers on graphite due to its 3D-structure. For statistical analysis of the coverage results, a 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 a two sided unequal variance 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 optimisation calculations were calculated where convergence tolerance regarding energy was 1.0e-4 kcal/mol, force was 0.005 kcal/mol/Å and 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 obtain better behaviour for the different stages of downstream minimization. Dynamic calculations were performed for an NVE ensemble with a temperature of 298 K, random values assigned for the initial velocities of the atoms and a time step of 0.1 fs 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 a 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 at some distance a chain of AMP molecules and finally two layers of a QAC crystal above the AMP chain. The entire system was filled with water molecules at 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 so 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 periodic structure of the QAC crystal layers. The deviation of the unit cell vectors of the QAC layers is 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 to be 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. The dimensions of the supercell are: plane x, y: 80.956 Å, 56.58 Å, and z: 103.4 Å.