Origins of the enhanced affinity of RNA-protein interactions triggered by RNA phosphorodithioate backbone modification


The well-characterized interaction between the MS2 coat protein and its cognate RNA hairpin was used to evaluate changes in affinity as a result of phosphorodithioate (PS2) replacing phosphate by biolayer interferometry (BLI). A structure-based analysis of the data provides insights into the origins of the enhanced affinity of RNA-protein interactions triggered by the PS2 moiety.

Selection of PD1/PD-L1 X-Aptamers


Specific, chemically modified aptamers (X-Aptamers) were identified against two immune checkpoint proteins, recombinant Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1). Selections were performed using a bead-based X-Aptamer (XA) library containing several different amino acid functional groups attached to dU at the 5-position. The binding affinities and specificities of the selected XA-PD1 and XA-PDL1 were validated by hPD-1 and hPD-L1 expression cells, as well as by binding to human pancreatic ductal adenocarcinoma tissue. The selected PD1 and PDL1 XAs can mimic antibody functions in in vitro assays.


X-Aptamer Selection and Validation


Aptamers and second generation analogs, such as X-Aptamers (XAs), SOMAmers, locked nucleic acids (LNAs), and others are increasingly being used for molecular pathway targeting, biomarker discovery, or disease diagnosis by interacting with protein targets on the surface of cells or in solution. Such targeting is being used for imaging, diagnostic evaluation, interference of protein function, or delivery of therapeutic agents. Selection of aptamers using the original SELEX method is cumbersome and time-consuming, often requiring 10–15 rounds of selection, and provides aptamers with a limited number of functional groups, namely four bases of DNA or RNA, although newer SELEX methods have increased this diversity. In contrast, X-Aptamers provide an unlimited number of functional groups and thus are superior targeting agents. Here, we discuss the X-Aptamer selection process.

RNA Nanostructures pp 151-174. Part of the Methods in Molecular Biology book series (MIMB, volume 1632). 21 July 2017.