Herein exploiting several spectroscopic techniques i e CD UV

Herein, exploiting several spectroscopic techniques, i.e. CD, UV and NMR, along with gel electrophoresis, size exclusion chromatography and in silico prediction analysis, the conformational behaviour of R1.2 and R1.3 was studied in Na+- and K+-rich solutions, which mimic the extra- and intracellular media. In addition, a buffer containing both K+ and Na+ ions, in which the aptamers had been originally selected [17], was also studied for comparison. Then, the ability of R1.2 and R1.3 to bind mIgM expressed in human lymphoma cells, BJAB, was investigated by flow cytometry assays, exploiting the 3′-FAM-labeled analogues of R1.2 and R1.3 to obtain the binding affinity data. Thus, to ensure that the FAM fluorescent tag had no relevant effect on R1.2 and R1.3 solution behaviour, the conformational properties of the labeled oligomers were studied in comparison with the unmodified aptamers. Overall, this study was aimed at evaluating the preferred secondary structures of R1.2 and R1.3 under different solution conditions, as well as their thermal stability. Particularly, the role of cations in regulating the aptamer polymorphism in solution was analysed. Interestingly, the binding affinity of R1.2 and R1.3 for mIgM was significantly enhanced in K+-rich solutions, suggesting that a G-quadruplex structuring is of crucial relevance in the aptamer-target recognition.
Results and discussion
Conclusions Structural elucidation of how aptamers fold in solution and bind to cell surface receptors is yet to be explored. There are few solved structures of aptamers bound to soluble Efavirenz sale available in the literature [49,50]. However, when the target of the studied aptamer is a receptor protein expressed on the cell surface, elucidation of the aptamer functional folding is nearly impossible. Here, we undertook a study to gain insight into the solution behaviour of two G-rich aptamers, termed R1.2 and R1.3, selected against mIgM expressed on BJAB cells. Using a combined approach involving several biophysical techniques and in silico prediction analysis, their conformational properties were investigated in different pseudo-physiological buffers, favouring either duplex or G-quadruplex structures. Conclusively, this study proved that both R1.2 and R1.3 are polymorphic aptamers, adopting mainly parallel, unimolecular G-quadruplex structures in K+-rich buffers, with specific features and thermal stability largely dependent on the solution conditions, as assessed by CD, CD-melting, UV, UV-melting, native PAGE and SE-HPLC analysis. Similar properties were displayed by their 3′-FAM-labeled analogues, here used in flow cytometry experiments to determine the ability of R1.2 and R1.3 to selectively recognize IgM proteins. Remarkably, affinity constants in the low nanomolar range were determined for both aptamers in the Tris/K+ buffer, while no binding with the protein was observed in the PBS/Mg2+−buffer. Under the latter conditions, the G-quadruplex structure of the aptamers is destabilized in favour of hairpin duplex-based architectures. On the other hand, the Tris/K+ buffer favours the G-quadruplex formation, indicating that both R1.2 and R1.3 fold into G-quadruplex structures in their bioactive conformations, which is necessary for a specific and high affinity recognition of mIgM expressed in B cells.
Methods
Acknowledgments This work was supported by the AIRC -Italian Association for Cancer Research (IG2015 n. 17037 to D.M.). We deeply thank prof. John O. Trent for freely providing us with the advanced software described in del Villar-Guerra et al. G-Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy. Angew. Chimie Int. Ed. 2018, 57, 7171-7175, allowing the deconvolution of the CD spectra and the evaluation of the relative contributions of parallel and antiparallel/hybrid G4s for both aptamers R1.2 and R1.3 under G4-forming conditions.