Malgorzata Sierant

Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection

Short interfering RNAs (siRNAs) are valuable reagents for sequence-specific inhibition of gene expression via the RNA interference (RNAi) pathway. Although it has been proposed that the relative thermodynamic stability at the 5′-ends of siRNAs plays a crucial role in siRNA strand selection, we demonstrate here that a character of the 2-nt 3′-overhang of siRNAs is the predominant determinant of which strand participates in the RNAi pathway. We show that siRNAs with a unilateral 2-nt 3′-overhang on the antisense strand are more effective than siRNAs with 3′-overhangs at both ends, due to preferential loading of the antisense strand into the RNA-induced silencing complex (RISC). Regardless of the relative thermodynamic stabilities at the ends of siRNAs, overhang-containing strands are predominantly selected as the guide strand; whereas, relative stability markedly influences opposite strand selection. Moreover, we show that sense strand modifications, such as deletions or DNA substitutions, of siRNAs with unilateral overhang on the antisense strand have no negative effect on the antisense strand selection, but may improve RNAi potency. Our findings provide useful guidelines for the design of potent siRNAs and contribute to understanding the crucial factors in determining strand selection in mammalian cells.

Evoking picomolar binding in RNA by a single phosphorodithioate linkage

RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ∼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference.

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimers disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

Crystal structure, stability and Ago2 affinity of phosphorodithioate-modified RNAs

Small interfering RNAs (siRNAs) with phosphorodithioate modifications (PS2-RNA) possess favourable properties for use as RNAi therapeutics. Beneficial here is the combining of PS2 and 2′-O-methyl modifications (MePS2). SiRNAs with MePS2 moieties in the sense strand show promising efficacies in vitro and in vivo. Crystal structures of PS2- and MePS2- modified RNAs reveal subtle changes in geometry and hydration compared with natural RNA. A model of an MePS2-RNA–PAZ domain complex points to a hydrophobic effect as the source of the higher affinity of MePS2-RNA for Ago2.

DNA binding and cleavage studies of copper(II) complexes with 2′-deoxyadenosine modified histidine moiety

This work is focused on the study of DNA binding and cleavage properties of 2′-deoxyadenosines modified with ester/amide of histidine (his6dA ester, his6dA amide) and their copper(II) complexes. To determine the coordination mode of the complex species potentiometric and spectroscopic (UV–visible, CD, EPR) studies have been performed. The analysis of electronic absorption and fluorescence spectra has been used to find the nature of the interactions between the compounds and calf thymus DNA (CT-DNA). There is significant influence of the –NH2 and –OCH3 groups on binding of the ligands or the complexes to DNA. Only amide derivative and its complex reveal intercalative ability. In the case of his6dA ester and Cu(II)–his6dA ester the main interactions can be groove binding. DNA cleavage activities of the compounds have been examined by gel electrophoresis. The copper complexes have promoted the cleavage of plasmid DNA, but none of the ligands exhibited any chemical nuclease activity. The application of different scavengers of reactive oxygen species provided a conclusion that DNA cleavage caused by copper complexes might occur via hydrolytic pathway.

Photosensitive nanocapsules for use in imaging from poly(styrene-co-divinylbenzene) cross-linked with coumarin derivatives.

The study objective was to generate biocompatible probes and develop a stable macromolecule imaging system that are based on nanolipopolymersomes and can be used in living cells. We synthesized nanolipopolymersomes with a fluorescent polymer wall surrounded by an outer phospholipid shell that exhibits potential for the controlled delivery of diagnostic agents to cells. We describe a new type of probe suitable for dual detection methods (spectrophotometric and fluorescence). This aspect makes it unique among currently available probes because allows it to be detected with greater accuracy. We developed a highly fluorescent coumarinated polymer to overcome the limited brightness of conventional dyes with insufficient for long-term photostablility. Hydrophilic dyes (Lucifer yellow, Procion red, Procion blue) are entrapped in the aqueous core of stable polymeric nanocapsules with coumarin 6 embedded in a nanometre-thick poly(styrene-co-divinylbenzene) wall. Target compounds can be incorporated into nanocapsules in a single step. The hydrophilic phospholipids outer shell ensures biocompatibility and facilitates cell penetration. In this way, the novel fluorescent hybrid materials can help of nanotechnology.

S-Geranyl-2-thiouridine wobble nucleosides of bacterial tRNAs; chemical and enzymatic synthesis of S-geranylated-RNAs and their physicochemical characterization

Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.

Nanocapsules for 5-fluorouracil delivery decorated with a poly(2-ethylhexyl methacrylate-co-7-(4-trifluoromethyl)coumarin acrylamide) cross-linked wall

The first synthesis of polymer vesicles with reverse cross-linked walls from poly[2-ethylhexylmethacrylate-co-(7-(4-trifluoromethyl)coumarin acrylamide)] P(EHMA/FMCA) is reported. The synthesis, carried out under UV light (λ = 350 nm), was achieved via radical photopolymerization of hydrophobic compounds saturating the bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome template. The polymerization reaction with coumarin moieties has a pivotal role, because the polymer chains become cross-linked as a result of the dimerization of the coumarin side chains of the formed polymers and, in this way, spherical capsules are obtained. Moreover, thanks to the incorporation of coumarin moieties, as an additional bonus, a nanometer-thick porous, fluorescent wall was formed, avoiding the use of additional cross-linking reagents, which might deactivate the encapsulated payloads. We present a new paradigm of release drug delivery systems combining a long shelf-life with the ability for rapid release using external factors such as light. To demonstrate this concept, target compounds such as hydrophilic dyes (Lucifer yellow (LY), phenosafranin (PhS)) and the drug 5-fluorouracil (5-FU)) were incorporated inside the stable, hollow, polymeric nanocapsules. The goal of these applications depends on the versatile strategy that is used for the preparation of highly emissive nanometer-sized polymer capsules with surface functionalities and optical properties for diagnostic applications.

Evaluation of BACE1 Silencing in Cellular Models

Beta-secretase (BACE1) is the major enzyme participating in generation of toxic amyloid-beta (Aβ) peptides, identified in amyloid plaques of Alzheimers disease (AD) brains. Its downregulation results in decreasing secretion of Aβ. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus) were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, βIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR), however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed.

RNA interference in silencing of genes of Alzheimer's disease in cellular and rat brain models

Accumulation of insoluble aggregates of beta-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimers disease. The major enzymes required for the generation of toxic amyloid-beta peptide are beta-(BACE1) and gamma-secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of gamma-secretase, responsible for development of Familial Alzheimers disease. Reducing the level of beta-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimers disease.