David John Clarke

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)₂Gly]₂ (BC–miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC–miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA. A thorough study of mono- and bis-BC–miRNases (containing one or two catalytic peptides, respectively) revealed that: (i) the sequence of miRNA bulge loops and neighbouring motifs are of fundamental importance for efficient miRNA cleavage (i.e., motifs containing repeating pyrimidine–A bonds are more susceptible to cleavage); (ii) the incorporation of the second catalytic peptide in the same molecular scaffold increases the potency of BC–miRNase, providing a complete degradation of miR-17 within 72 h; (iii) the synergetic co-operation of BC–miRNases with RNase H accelerates the rate of miRNA catalytic cleavage by both the conjugate and the enzyme. Such synergy allows the rapid destruction of constantly emerging miRNA to maintain sufficient knockdown and achieve a desired therapeutic effect.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge–loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge–loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

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⁺), 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 non-physiological conditions did conjugates form strong complexes with non-complementary RNAs.

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)₂Gly]₂ capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 10³ miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide build-ing blocks to provide sequence-specific, PCR-free fluorescent detection of un-labelled nucleic acid sequences. We discrimi-nated at picomolar detection limits (<7 pM) ‘perfect-match’ from mismatched sequences, down to a single nucleotide muta-tion, buried within longer lengths of target. Detailed characterisation by NMR, TEM, mass spectrometry and rheology pro-vided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sen-sitivity and detection limit. We discuss the generic design, which is based on a highly-predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g. SNPs) and RNA (messenger, micro and viral genomic RNA).

Contradictions in the reported biocompatibility of graphene-related materials have been attributed to differences in their preparation. Herein, we address the conflicting behavior of different pristine graphene dispersions through their careful preparation and characterization in aqueous media. Although exfoliated in different media, all graphene dispersions were physically similar and comprised few-layer graphene flakes of 100 to 400 nm mean length with relatively defect-free basal planes. The dispersions were colloidally stable, including in physiological saline and when organic solvents were exchanged with water by dialysis, due to their negative zeta potentials (−28 mV to −60 mV) from interaction with water and the different dispersants. Thus, we have been able to establish the influence of pre-association with different dispersants, including those likely to be encountered during the transport and excretion of graphene in vivo. Shear-forced association with neutral phospholipids was transient, as the lipids desorbed to form liposomes, and left a hemolytic dispersion, whereas other dispersions were not hemolytic. High boiling point solvents widely used to exfoliate graphene are toxic and viewed difficult to remove, but were readily removed by simple dialysis. Human serum albumin readily and stably adsorbed in predominantly monomeric form to pristine graphene in physiological saline, which may be expected in vivo. This work shows the large influence that different adsorbates can have on the behaviour of otherwise physically-similar graphene.

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at “proof of concept” level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent andMDR1-transfected Madin–Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose–response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.