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DNA, Environment, Mineral Association: Reaction Kinetics

Periodic Reporting for period 1 - DENMARK (DNA, Environment, Mineral Association: Reaction Kinetics)

Reporting period: 2020-04-01 to 2022-03-31

Traces of DNA can be released into the environment through faeces, urine or decomposition of dead organisms. Such environmental DNA can provide valuable information on past and present organisms. Recovering eDNA can be extremely difficult as biotic (e.g. microbial attack) and/or abiotic (e.g. pH, temperature, hydrolysis) factors can lead to its partial or complete destruction. However, adsorption of DNA molecules onto the surfaces of minerals may significantly decrease the DNA decay rate. Understanding the DNA-mineral interaction is key both for accessing the mechanisms controlling the DNA-mineral association and understanding the longevity of sediment preserved eDNA.

Therefore, the aim of DENMARK is to:
1. assess different DNA-mineral associations and eDNA preservation state (concentration and damage patterns) in sediments to explore the longevity of each DNA-mineral system.
2. investigate the in vitro DNA-mineral interactions at the single bond level (bond rupture forces) in different environmentally relevant solution compositions to quantify the energy and kinetic parameters associated with the interacting bonds.
3. examine the in vitro DNA-mineral interactions at the bulk level (nature, fraction and stability of interacting bonds), and link those to the single bond level energetics to quantify the longevity of the DNA-mineral bonds in different environmentally relevant solution compositions.
4. link the sediment data to the in vitro data to make a conceptual model for addressing the characteristics and longevity of each DNA-mineral system, and improve eDNA sampling, extraction and analysis protocols.

Overall, our data suggest that the DNA mineral association (adsorption and desorption) is strongly influenced by the interfacial geochemistry, and it is sensitive to the mineral surface charge, mineral charge density, solution composition, salinity, and pH.
RO1
The aim of RO1 was to assess the relationship between sediment geochemistry and DNA concentration to better understand the longevity of eDNA in different mineral systems.
Work performed and Results: I have carried out statistical analysis on 62 samples and 15 variables (XRF data) from 4 sediment cores (Spring Lake, Charlie Lake, Dragons Hollow, and Haväng) with different mineralogical characteristics and chronologies (13 kyr to present). The principal component analysis revealed three main groups in our dataset (Fig.1; Table 1). The chemical elements of the large group (Fe, K, Ti, Si, Rb, Ar, Zr, Ni, Mn, Al) show high positive loadings on PC1 and generally low negative loadings on PC2. The second group (Ca, Sr) exhibits moderate positive scores on PC1 and strong positive loadings on PC2, while group 3 (DNA (ng/μL) and Br) shows slightly negative loadings on PC1 and strong positive scores on PC2. Thus looking at the axes and the loadings it is clear that the DNA concentration (ng/μL) shows a strong linear correlation with Br, a weak/moderate positive correlation with Ca and Sr, and no correlation with any of the other chemical elements of group 1. The sediments from Spring Lake generally contain higher amounts of DNA (mean=9.1±8.7 ng/μL), whereas Charlie Lake sediments (mean=0.78±0.7 ng/μL) the lowest (Games-Howell p≤0.01). Interestingly, Ca, Sr and Br are all significantly higher in the Spring Lake sediments (Table 2).

RO2
The purpose of RO2 was to visualise DNA adsorption on different mineral surfaces, and quantify the rupture forces (bond breaking) using an atomic force microscope (AFM).
Work performed and Results: I used calcite and hematite crystals, different single and double stranded DNA sequences, and different solution compositions to conduct the AFM experiments (tapping, contact force and dynamic force modes). Our data indicate that adsorption of ssDNA (Fig.3a-c) and dsDNA (Fig.3d-e) to calcite surfaces (terraces and step edges) is affected by the mineral surface topography and the ionic strength of the salts (MgCl2; NaCl). Plasmid DNA (Fig.3f) only adsorbs to the step edges which carry more positive charge than the terraces. The data highlights that in most cases the ssDNA has an increased affinity to the terrace than ds. The adsorption of ssDNA and plasmid (DNA appears as bright features) onto calcite (Fig.4a-c) and hematite (Fig.4d-f) surfaces is clearly different and affected by the surface topography. On hematite crystals (MgCl2, NaCl or no ions in solution), no ssDNA adsorption can be observed, whereas plasmid adsorbs onto hematite surfaces. Rupture forces are also significantly different between calcite and hematite surfaces, and are also affected by the salts and their concentration in the solution (Fig.5). The MgCl2 solution creates a higher ionic strength than the NaCl solution because of the double charge on the Mg. A higher ionic strength causes a smaller electrical double layer enabling a higher adsorption strength.

RO3
The aim of RO3 was to examine the DNA-mineral interactions at the bulk level (nature, fraction and stability of interacting bonds) and link those to the AFM data (RO2) to understand the longevity of the DNA-mineral bonds in different environmentally relevant solution compositions (RO4).
Work performed and Results: I used surface sensitive Fourier transform infrared spectroscopy in attenuated total reflectance (ssFTIR-ATR). I measured both the DNA adsorption and desorption kinetics for goethite (iron oxide; same family with hematite from WP2). Our results show that the DNA binds to goethite mainly through cationic bridging with the sugar-phosphate DNA backbone, but also with some minor direct interactions with the DNA nitrogenous bases (Fig.6 and Fig.7). DNA adsorption was significantly affected by the background electrolytes (type and concentration) and pH. Similarly, desorption of DNA from the goethite film was promoted by increased salt concentration and changes in the pH (Fig.8).

RO4
The aim of RO4 was to link the sediment to the in vitro data to make a conceptual model for addressing the characteristics and longevity of each DNA-mineral system.
Work performed and Results: From RO2 and RO3 it is clear that the DNA mineral association is strongly influenced by interfacial geochemistry and is sensitive to mineral surface charge, mineral charge density, pH, solution composition and salinity. The AFM images reveal the DNA adsorption behaviour (conformation and sites for adsorption) changes with the background cations. The FTIR experiments show that a cation with a high ionic potential is able to immobilise the DNA on a negatively charged surface, whereas the DNA molecule would move around on the surface with the background solution only containing cations with a low ionic potential. The sensitivity of the DNA-mineral bond to solution composition (background electrolytes, pH) also influences desorption kinetics, and hence longevity of the DNA-mineral association through time and space.
Data generated by DENMARK can advance existing eDNA extraction and analysis protocols, and could be exploited by various organisations for the identification of the presence of endangered or invasive species that is very difficult to detect visually. Researchers working in the fields of biodiversity, environmental and biomedical science or relevant fields will also benefit from such an advancement of extraction and analysis techniques.
WP1 results
WP2 results
WP2 results
WP1 results
WP1 results
WP3 results
WP3 results
WP3 results
WP2 results
WP1 results