Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput affinity screening.

We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA--ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel.

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

The effect of backbone charge on the collision-induced dissociation of oligonucleotides.

Knowledge of the effects of structural changes in oligonucleotides on their dissociation reaction is important in the application of mass spectrometry to sequence determination. The effect of backbone charge on the collision-induced dissociation of multiply-charged oligonucleotides produced by electrospray was explored by examination of models in which the normal phosphodiester linkage was partially replaced with an uncharged methylphosphonate (MP) linkage. Three different MP-containing oligonucleotides were studied, designed to represent a concentration of charge on the 5'- and 3'-ends of the molecule and with an even distribution of charge along the backbone, compared with a control molecule containing only phosphodiester linkages. In all MP-containing oligonucleotides charging of over 90% of phosphate groups were observed, compared with typical charging patterns of about 60% in normal all-phosphodiester oligonucleotides. This unexpected effect is attributed to charge stabilization by interactions of charged sites with uncharged residues. Analysis of the collision-induced dissociation mass spectra showed that backbone cleavage occurred at every residue (w and a-base ion series), producing a full set of sequencing ions whether or not the linkage at that site was formally charged. It is concluded that under the multiple collision conditions of the quadrupole collision cell that backbone cleavage proceeds through two generic pathways, one involving base loss followed by cleavage of the adjacent C3'-CO bond and the other requiring neither base loss nor charged phosphate at the cleavage site. These results suggest that backbone cleavage reactions in conventional phosphodiester oligonucleotides can occur at non-ionized linkage sites, of which there are a high proportion in both electrospray- and MALDI-produced molecular ions.