Synthesis and Structural characterization of selex-derived rna aptamers by multidimensional nmr spectroscopy

The last few decades have seen an increased focus on the development of new instruments for the advancement of diagnostic assays. The monoclonal antibodies have always been considered the best candidates in the research for strategies with augmented sensitivity and selectivity. In recent years, molecular recognition systems based on RNA and DNA oligomers (aptamers) have emerged as novel molecular tools, useful for basic and applied aspects of molecular medicine. These have proved to be a valid altemative to antibodies or to other biosensors, in both therapeutic and diagnostic applications. In fact, they pose many advantages, such as heat stability, high tolerance over a wide range of pH and ionic strength conditions, as well as the fact that they can be synthesized by chemical methods. The PCR-based techniques (SELEX, systematic Evolution of Ligands by Exponential Enrichment) starting from random libraries of short ribonucleotide segments, allow the isolation of sequences with huge moìecular recognition capabilities and association constants (10^9 M^-1), making feasibìe the analysis of pico- or even femtomoles of substance. Recently, aptamers for the scrapie-sensitive ovine prion protein have been developed starting from 2'-fluorinated RNA-oligomers, which improve resistance to RNase. Two sequences (29 and 38 nucleotides each) have been identified constituting the minimum elements of molecular recognition of the ovine prion protein (Kd=14.6nM). Despite the great interest in this type of molecular recognition systems, nothing bas been done on the three-dimensional structure of the isolated aptamers. In this work we present the synthesis on solid phase and a preliminary structural characterization of both aptamers (APTA-38 and APTA-29) and related fragments (APTA-25 and APTA-19) by multidimensional mono and heteronuclear NMR experiments performed in H2O/D2O (9:l) and in D2O. The equilibrium haìrpin-double strand has been studied by DOSY experiments.