Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics.

When used as inhibitors of gene expression in vivo, oligonucleotides require modification of their structures to boost their binding affinity for complementary target RNAs. To date, hundreds of modifications have been designed and tested but few have proven to be useful. Among those investigated are mono- and polyamino-groups. These are positively charged at physiological pH and have been appended to oligonucleotides in an effort to reduce electrostatic repulsion during hybridization to RNAs, but have generally shown relatively minor benefits to binding. We conjugated spermine to uracils in oligonucleotides via a triazole linker so that the polyamine fits in the major groove of a subsequently formed RNA-duplex. The modifications produced large increases in target-binding affinity of the oligonucleotides. Using surface plasmon resonance-based assays, we showed that the increases derived mainly from faster annealing (kon ). We propose that the spermine fragments play a similar role to that of natural polyamines during oligonucleotide-target interactions in cells, and may be advantageous for oligonucleotides that operate catalytic mechanisms.

Site-Specific Labeling of MicroRNA Precursors: A Structure-Activity Relationship Study.

Functionalized oligoribonucleotides are essential tools in RNA chemical biology. Various synthetic routes have been developed over recent years to conjugate functional groups to oligoribonucleotides. However, the presence of the functional group on the oligoribonucleotide backbone can lead to partial or total loss of biological function. The limited knowledge concerning the positioning of functional groups therefore represents a hurdle for the development of oligoribonucleotide chemical tools. Here we describe a systematic investigation of site-specific labeling of pre-miRNAs to identify positions for the incorporation of functional groups, in order not to hinder their processing into active mature miRNAs.

A Small-Molecule Inhibitor of Lin28.

New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs. Here, we describe a protein/RNA FRET assay using a GFP-Lin28 donor and a black-hole quencher (BHQ)-labeled let-7 acceptor, a fluorescent protein/quencher combination which is rarely used in screening despite favorable spectral properties. We tested 16 000 molecules and identified N-methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide, which blocked the Lin28/let-7 interaction, rescued let-7 processing and function in Lin28-expressing cancer cells, induced differentiation of mouse embryonic stem cells, and reduced tumor-sphere formation by 22Rv1 and Huh7 cells. A biotinylated derivative captured Lin28 from cell lysates consistent with an on-target mechanism in cells, though the compound also showed some activity against bromodomains in selectivity assays. The Lin28/let-7 axis is presently of high interest not only for its role as a bistable switch in stem-cell biology but also because of its prominent roles in numerous diseases. We anticipate that much can be learned from the use of this first reported small molecule antagonist of Lin28, including the potential of the Lin28/let-7 interaction as a new drug target for selected cancers. Furthermore, this approach to assay development may be used to identify antagonists of other RBP/RNA interactions suspected to be operative in pathophysiological mechanisms.

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.

Polyamine-oligonucleotide conjugates: a promising direction for nucleic acid tools and therapeutics.

Chemical modification and/or the conjugation of small functional molecules to oligonucleotides have significantly improved their biological and biophysical properties, addressing issues such as poor cell penetration, stability to nucleases and low affinity for their targets. Here, the authors review the literature reporting on the biophysical, biochemical and biological properties of one particular class of modification - polyamine-oligonucleotide conjugates. Naturally derived and synthetic polyamines have been grafted onto a variety of oligonucleotide formats, including antisense oligonucleotides and siRNAs. In many cases this has had beneficial effects on their properties such as target hybridization, nuclease resistance, cellular uptake and activity. Polyamine-oligonucleotide conjugation, therefore, represents a promising direction for the further development of oligonucleotide-based therapeutics and tools.

Properties of N(4)-methylated cytidines in miRNA mimics.

Experiments conducted with micro RNA (miRNA) mimics often result in subtle phenotypic changes and hence require careful controls. A commonly used type of control reagent in the antisense/RNA interference fields is the mismatched sequence. However, it is difficult to use mismatch controls for miRNAs, mainly because base permutation in the seed region may generate a new miRNA seed with its own associated target transcripts. We incorporated N(4)-methylcytidine and N(4),N(4)-dimethylcytidine into a series of RNAs using the convertible nucleoside approach and measured their effects on hybridization affinity with complementary RNAs, and on miRNA-mediated and small interfering RNA (SiRNA)-mediated silencing. We report here that incorporation of a single N(4),N(4)-dimethylcytidine into the seed region of miRNAs can be used as a new class of negative miRNA control which (1) does not constitute a new seed sequence; (2) is accepted by the RNA-induced silencing complex (RISC); (3) causes a significant loss of binding affinity to target RNAs; and (4) is synthesized conveniently into oligoribonucleotides.