Detection of nucleic acids and other low abundance components in native bone and osteosarcoma extracellular matrix by isotope enrichment and DNP-enhanced NMR

Sensitivity enhancement by isotope enrichment and DNP NMR enables detection of minor but biologically relevant species in native intact bone, including nucleic acids, choline from phospholipid headgroups, and histidinyl and hydroxylysyl groups. Labelled matrix from the aggressive osteosarcoma K7M2 cell line confirms the assignments of nucleic acid signals arising from purine, pyrimidine, ribose, and deoxyribose species. Detection of these species is an important and necessary step in elucidating the atomic level structural basis of their functions in intact tissue.

The extracellular matrix (ECM) is a complex, heterogeneous, and active, environment, playing structural roles, but also strongly inuencing cell behaviour in both homeostasis and pathology. The difficulty of understanding the composition and regulation of the ECM parallels the situation within cells, but with the added complication that many molecular components are insoluble. Solid-state NMR (ssNMR) can contribute to a better understanding of the ECM through direct detection of sparse species, made possible by a strategy of isotopic enrichment of native biomaterials 1 combined with rapidly evolving dynamic nuclear polarization (DNP) enhancement techniques. 2 Briey these are based on the transfer of the much greater electron spin polarization, excited by a microwave laser at the electron resonance frequency of an organic free radical dopant, to the (NMR active) nuclei of the molecules of interest, at low temperatures to maximize polarization. The conversion of electron to nuclear polarization results in NMR sensitivity enhancements of up to several orders of magnitude. Recently we have reported direct observation of important low abundance collagen post-translational modications (PTMs) in intact native, and model, ECM. 3 While we focussed on species derived from one specic amino acid (Lys) component of the ECM, it is also of great potential importance to investigate whether other sparse ECM species become observable with DNP enhancement. Non-protein minor species, besides low abundance amino acids, are of particular interest since they represent a realm in which NMR can provide atomic length scale molecular structural information complementing other current biophysical techniques. Incorporating non-protein molecules into the overall structural biology picture will broaden conceptions of which ECM components to investigate in further detail towards an integrated model of the biological and material properties of tissues.
Here, we report DNP-enhanced NMR on mouse bone 1 enriched in a wide range of 13 C, 15 N-amino acids. Where necessary to distinguish between low abundance protein, and non-protein, signals, we conrmed amino acid type assignments by comparison with ECM produced by cells cultured on media supplemented with one or a few specic amino acids, using methods already described. 4,5 Our previous analysis of bone focussed on the relatively abundant species observable with conventional ssNMR. The enhancement provided by DNP now reveals non-protein biomolecules biosynthesized from isotopically enriched amino acids and/or sugars, and their incorporation into bone. The preparation of stable isotope enriched biomaterials, and our DNP NMR methodology, is described in detail in the ESI, † Materials and methods sections.
We begin our investigation on bone with a suite of ssNMR characterizations under DNP signal enhancement conditions. A full 1D 15 N DNP NMR spectrum is shown in ESI Fig. S1. † Apart from the predictable major signals (from backbone and sidechain amides, and abundant Lys and Arg) the sensitivity enhancement of enrichment-DNP reveals a number of more minor resonances (Fig. 1A) from lower abundance species; the shis of many of those at high spectral frequency coincide closely with the common (deoxy)nucleotide base ring nitrogens (Biological Magnetic Resonance Database, BMRB, as summarized in ESI Schemes S1 † (DNA) and S2 (RNA)). Signals from the nitrogen atoms of protonated His imidazolium (ca. 180 ppm) and unprotonated imidazole (ca. 250 ppm) ring forms are also observed. 6,7 The His assignments are reinforced by 15  . The PC ethanolamine substructure is biosynthesized from a single serine unit so, in our bone material, all atoms will be concurrently labelled. Finally Fig. 1C also shows the distinctive Hyl N z -C d ngerprint correlations; the close correspondence with (U-13 C 6 , 15 N 2 )-Lys enriched ECM from vascular smooth muscle cells 3 is shown in ESI Fig. S4, † proving that our labelling -DNP NMR combination readily detects the Hyl PTM even in this complex organic-inorganic composite native biomaterial, as well as in simpler native (skin) and model ECMs. 3 Chemical shis are summarized in Table 1.
Detection of His, lipid components, and Hyl, in native biomaterials is not unexpected. In contrast nucleic acids in bone have been little discussed, although this tissue is a favourable source of forensic genetic material. 9 Given this underrepresentation of bone in nucleic acid literature, we turned to ECM from the aggressively metastasizing K7M2 osteosarcoma cell line 10 to substantiate these signals. A 13 C DNP NMR spectrum of K7M2 ECM supplemented with (U-13 C 2 , 15 N)-Gly (Gly*) and (U-13 C 6 )-glucose (Glc*) is shown in Fig. 2A; it shows a number of clear signals with shis consistent with the common (deoxy)nucleotide bases 11 (ESI Schemes S1 and S2 †). Abundant nucleic acid in the K7M2 ECM is conrmed by conventional in situ propidium iodide staining and confocal uorescence microscopy (ESI Fig. S5 †). The biosynthetic routes to the bases (ESI Scheme S3 †) dictates that all the base carbon atoms will be at least partially 13 C enriched by Gly* and Glc*, although only the purine C 4 -C 5 -N 7 substructure (from intact Gly*), and to some extent pyrimidine C 4 -C 5 -C 6 from Glc* via Ser, 12 will necessarily be concurrently labelled. (Deoxy)riboses are biosynthesized from Glc* and so all (deoxy)sugar rings will be concurrently 13 C enriched. Fig. 2B details results of a through-bond (J-transmitted) INADEQUATE, 13 which clearly shows the predicted purine C 4 -C 5 and pyrimidine C 4 -C 5 cross peaks. DARR 14 (Fig. 2C) conrms these and indicates pyrimidine C 6 signals in Cyt and Thy, and Ura from RNA. The presence of both DNA dRib, and RNA Rib, is conrmed by the low frequency part of the INADEQUATE spectrum (Fig. 2D). 15   NMR shows a clear signal from purine N 7 's, corresponding closely to that of the putative purine N 7 's in bone (Fig. 2E). Also a 13 C{ 15 N} TEDOR 15 (Fig. 2F) mapping short carbon-nitrogen distances, shows the expected purine N 7 -C 5 and weaker N 7 -C 4 correlations, but also purine N 7 -C 8 cross peaks (the latter atom acquired from Glc* via N-formyl tetrahydrofolate). All DNA and RNA shis measured in K7M2 ECM are summarized in Table 2.

N DNP
There are large number of catalogued nucleic acid shis in the BMRB but as yet no robust equivalent of the chemical shi index 16 of protein secondary structural elements, especially with respect to 15 N. Nucleotide base 13 C and 15 N shis are particularly sensitive to secondary structural features 17-20 but linewidths and overlap in our 13 C and 15 N spectra currently preclude structural conclusions about K7M2 ECM. In principle though this work suggests that the potential of 15 N shis as indices of nucleic acid structure 20,21 in whole tissues can be realized through several conformational "reporter" nitrogen atoms in the well resolved high frequency NMR window above 150 ppm. Nucleic acid in bone may be predominantly intracellular, unlike that of K7M2 where a higher extracellular proportion is expected, but it is interesting that these distinctive 15 N signals can be observed in both systems. We envisage that detection by DNP-NMR in native tissue is an essential step in fullling this potential to characterize nucleic acid structure in the intact tissue and ECM environment.
NMR detection of nucleic acids in bone raises fresh questions about their biological role, and avenues to new insights. NMR detection is an essential rst step in elucidating these roles at the molecular and atomic level in native materials. Extracellular roles e.g. signalling, of nucleic acids are likely to operate in K7M2 ECM, and potentially also in bone and bone diseases e.g. osteoporosis. 22 Bone undergoes constant remodelling so a high degree of transcriptional, translational, and apoptotic activities are expected, 23 leading to signicant amounts of nucleic acid being produced in bone cells and released in the ECM. Once in the extracellular environment, some nucleic acids may have a different function in addition to their intracellular roles. For instance, nucleic acids have pronounced affinity for apatite mineral, 24 and may participate in biomineralization, 25 as calcium sequestering and mineral templating scaffolds, a role that is already proposed for structurally related polynucleotides like poly-ADP-ribose. 4 Phospholipids 26 are abundant in mineralized bone. 27 They may represent the presence of matrix vesiclesextracellular structures ca. 100 nm in size, "pinched off" from the outer membranes of osteoblastswhich act as a nidus for initial  calcium phosphate precipitation in normal 28 and pathological 29 biomineralization. The molecular mechanisms underlying the initial bioprecipitation events and processes remain to be elucidated, in particular the role of matrix vesicle phospholipids. Direct observation of the PC headgroup by NMR in healthy bone is a signicant step in atomic level understanding of the relevant intermolecular and interionic interactions. In addition to matrix vesicles, of 40-100 nm sized exosomes are the basis of a pathway for cancer metastasis 30 and therapy resistance 31 through delivery of proteins, lipids, RNA and DNA to remote cells. 32 The cargo carried by these vesicles may play unexpected roles if released directly in the extracellular space and allowed to interact with other components of the ECM. 33 DNP-enhanced NMR is potential non-targeted method of better understanding the role played by these extracellular vesicles.
Hyl and its subsequent modications are essential to the structure, properties, and functions, of collagen. 34 The type I collagen heterotrimer contains 12 His residues. Both of those in each a1 (I) chain are two residues from those Lys residues which are sequence specically d-hydroxylated to Hyl, in a strongly conserved Lys-Gly-His-Arg tetrad. 35 These His residues have been proposed to play a part in targeting lysyl hydroxylases and thereby the formation of the specic Lys and Hyl crosslinks 36 essential to maintaining collagen and tissue structure. Some of the same His residues are not only hydroxylated to Hyl, but of these some are subsequently also enzymatically glycosylated to (Glc)GalHyl, an essential event in directing the as-yet poorly understood process of collagen bril assembly. It has recently been proposed that these self same residues are also preferentially glycated in non-enzymatic, usually harmful, reactions with metabolic sugars. 37 Finally there is evidence for a trivalent crosslink, histidinohydroxylysinonorleucine, between these same Lys/Hyl and His residues. 38 Thus His and Lys/Hyl residues co-exist in crosslinking and glycosylation/glycation "hotspots" which may be key to better understanding the spatial relationships underlying orderly, and pathological, collagen assembly and structure. Detecting His, Lys and Hyl simultaneously, as enabled by isotopic enrichment and DNP-enhanced NMR, in native and model ECM, opens fertile new possibilities for studying these processes.
This communication demonstrates again 3 the ability of ssNMR, combined with signal enhancement by DNP and targeted isotope labelling, to detect low abundance components in intact tissue that may have surprising biological roles, thus complementing other biophysical and molecular biology techniques in exploring diverse areas of matrix biology.

Conflicts of interest
There are no conicts of interest. Animal procedures were unregulated in terms of the U. K. Animals (Scientic Procedures) Act 1986, but nevertheless were reviewed for compliance with institutional ethical guidelines by the Animal Welfare Ethical Review Board, University of Cambridge.