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

Abstract

Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. ‘Smart’ supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-β anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an ‘in-line’ attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

“…Historically, the first method applied for the synthesis of ss-aRNase was a fragment conjugation in solution, when the individual structural components (i.e., an oligonucleotide and a catalytic moiety), which were separately synthesised, deprotected and isolated, were then allowed to react with each other in the presence of respective condensing or activation reagents [ 43 , 44 , 45 , 46 ]. Nowadays, this approach has been successfully applied for the synthesis of peptidyl-oligonucleotide conjugates of various design [ 34 , 36 , 42 , 47 , 48 , 49 , 50 ]. Another version of fragment conjugation, which was widely applied for the synthesis of various ss-aRNases, was also based on the post-synthetic coupling between the key players, when one of the reacting components (usually oligonucleotide) was still bound to the solid support, while the second component (usually catalytic moiety) was in solution [ 26 , 30 , 31 , 32 , 37 , 38 , 40 , 51 , 52 , 53 ].…”

“…The synthesis of “single” [ 34 , 35 , 36 , 50 ], “hairpin” [ 29 , 30 , 44 ], “dual” [ 47 ] and “bulge-inducing” [ 42 ] peptidyl-oligonucleotide conjugates (POCs) was carried out using fragment conjugation in solution. The synthetic peptide was attached to either one (in the case of “singe”, “hairpin” or “bulge-inducing” POCs) or two oligonucleotide recognition motifs (in the case of “dual” conjugates) in DMSO, which often required the use of a DMSO-soluble cetyltrimethylammonium salt of the appropriate oligonucleotide(s).…”

“…Peptidyl-oligonucleotide conjugates (POCs) represent another large class of ss-aRNases, which are based on incorporation of the catalytic peptide into the oligonucleotide recognition motif(s) at different positions using a broad variety of attachment methods [ 34 , 35 , 36 , 42 , 47 , 50 ]. Among others, peptides with alternating leucine and arginine residues [(LeuArg) 2 Gly] 2 and [(LeuArg) 4 Gly] proved to be the most efficient cleavers as part of the ss-aRNase subject to the certain rules of conjugate design.…”

“…Indeed, the conjugates with an aminohexyl linker attached at the C1′ position of the anomeric sugar in either the α- or β-configuration appeared to be effective cleavers (with half-life for target RNA of 8 h). In contrast, the attachment of the same peptide to the aromatic ring (e.g., C8-position of adenosine) showed zero cleavage activity [ 42 ].…”

“…Presumably, the reduced affinity of the aRNase to the target after each cleavage event facilitates the release of ss-aRNase from the hybridised complex. Such hypotheses turned out to be rather successful and became widely used for the creation of several types of ss-aRNases incorporating different catalytic moieties, some of them demonstrating catalytic turnover [ 30 , 37 , 42 ].…”