1H NMR spectroscopy quantifies visibility of lipoproteins, subclasses, and lipids at varied temperatures and pressures.

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.

Fluorescent hydrophobic zippers inside duplex DNA: interstrand stacking of perylene-3,4:9,10-tetracarboxylic acid bisimides as artificial DNA base dyes.

Perylene-3,4:9,10-tetracarboxylic acid bisimides (PBs) were incorporated synthetically into oligonucleotides by using automated DNA building-block chemistry. The 2'-deoxyribofuranoside of the natural nucleosides was replaced by (S)-aminopropan-2,3-diol as an acyclic linker between the phosphodiester bridges that is tethered to one of the imide nitrogen atoms of the PB dye. The S configuration of this linker was chosen to mimic the stereochemical situation at the 3'-position of the natural 2'-deoxyribofuranosides. By using this strategy, up to six PB dyes were incorporated in the middle of 18-mer DNA duplexes by using interstrand alternating sequences of PBs with thymines or an abasic site analogue. Both PB dimers and PB hexamers as artificial base substitutions inside the duplexes yield characteristic excimer-type fluorescence. The stacking properties of the PB chromophores are modulated by the presence or absence of thymines opposite the PB modification site in the counterstrand. The interstrand PB dimers can be regarded as hydrophobically interacting base pairs, which display a characteristic fluorescence readout signal. Hence, for the PB hexamers, we proposed a zipperlike recognition motif that is formed inside duplex DNA. The PB zipper shows characteristic excimer-type emission as a fluorescence readout signal for the pairing interaction.