RNA knockdown by synthetic peptidyl-oligonucleotide ribonucleases: behavior of recognition and cleavage elements under physiological conditions

Abstract

Sequence-specific protein-based ribonucleases are not found in nature. Absolute sequence selectivity in RNA cleavage in vivo normally requires multi-component complexes that recruit a guide RNA or DNA for target recognition and a protein-RNA assembly for catalytic functioning (e.g. RNAi molecular machinery, RNase H). Recently discovered peptidyl-oligonucleotide synthetic ribonucleases selectively knock down pathogenic RNAs by irreversible cleavage to offer unprecedented opportunities for control of disease-relevant RNA. Understanding how to increase their potency, selectivity and catalytic turnover will open the translational pathway to successful therapeutics. Yet, very little is known about how these chemical ribonucleases bind, cleave and leave their target. Rational design awaits this understanding in order to control therapy, particularly how to overcome the trade-off between sequence specificity and potency through catalytic turnover. We illuminate this here by characterizing the interactions of these chemical RNases with both complementary and non-complementary RNAs using T m profiles, fluorescence, UV-visible and NMR spectroscopies. Crucially, the level of counter cations, which are tightly-controlled within cellular compartments, also controlled these interactions. The oligonucleotide component dominated interaction between conjugates and complementary targets in the presence of physiological levels of counter cations (K þ), sufficient to prevent repulsion between the complementary nucleic acid strands to allow Watson-Crick hydrogen bonding. In contrast, the positively-charged catalytic peptide interacted poorly with target RNA, when counter cations similarly screened the negatively-charged sugar-phosphate RNA backbones. The peptide only became the key player, when counter cations were insufficient for charge screening; moreover, only under such nonphysiological conditions did conjugates form strong complexes with non-complementary RNAs.

“Bind, cleave and leave”: multiple turnover catalysis of RNA cleavage by bulge–loop inducing supramolecular conjugates

“…The extra peptide chain seemed to stabilize the hybridized complex, presumably through additional electrostatic interactions between the negatively-charged RNA chain and the positively-charged peptide. Such a stabilizing effect was also observed for the previously-reported linear conjugates with the 5’-terminal attachment of the amphiphilic peptides ( 76 ). The difference in the association constants between BC5-L-αα, BC5-L-αβ and BC5-L-βα did not exceed statistical errors (Table 1 ).…”

“…Indeed, multiple-turnover catalysis usually requires a significant excess of RNA substrates (up to 30-fold) over the catalytic molecules, thus resulting in a high concentration of RNA sequences in solution. This may trigger some level of substrate aggregation, which can be further facilitated by formation of multiple peptide bridges between different RNA-conjugate hybrids, stabilized by electrostatic interactions between the positively-charged peptides and the negatively-charged sugar-phosphate backbones of nucleic acids ( 76 ). Consequently, in order to study reaction kinetics in a comparable manner to enzymes, we devised a simple, linear fluorescently-labelled RNA lacking such secondary and tertiary structure, so that supramolecular RNA structure did not dominate the kinetics.…”

Dual miRNases for Triple Incision of miRNA Target: Design Concept and Catalytic Performance

“…The amphipathic nature of the peptide component within the conjugates, and the fact that each conjugate contains both positively-charged catalytic peptide and negatively-charged oligonucleotides, may suggest that such structures may potentially form inter-molecular assemblies. Indeed, we have recently shown by NMR Diffusion Ordered Spectroscopy [55] that in de-ionized water (e.g., in the absence of counter ions) the short "single" peptidyl-oligonucleotide conjugates may self-assemble into dimers, presumably via electrostatic interactions between the positively-charged peptide of one molecule and the negatively-charged oligonucleotide of a neighboring molecule. However, in contrast to the aggregation downsides reported for PNA conjugates, such self-assembly of the peptidyl-oligonucleotide conjugates into supra-molecular structures might be beneficial as a key factor contributing to catalysis.…”

Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates

“…ESI mass spectral data was collected on a Thermo Scientific LTQ Orbitrap XL (MA, USA) at the EPSRC UK National Mass Spectrometry Facility at Swansea University (Swansea, UK). MALDI (Matrix-Assisted Laser Desorption Ionisation) mass spectra was collected on a Bruker Dal-tonics Ultraflex TOF/TOF mass spectrometer (MA, USA) at the Manchester Interdisciplinary Biocentre, University of Manchester as described earlier ( 34 ). 1 H NMR spectra were recorded using Bruker Avance II+ spectrometers operating at proton frequencies of 400 MHz using BBI 1 H/D-BB Z-GRD Z8202/0347 probe using previously described methods ( 34 , 35 ).…”