Short RNA Sequences Research Articles

ShRNA-seq data analysis with edgeR

Pooled short hairpin RNA sequencing (shRNA-seq) screens are becoming increasingly popular in functional genomics research, and there is a need to establish optimal analysis tools to handle such data. Our open-source shRNA processing pipeline in edgeR provides a complete analysis solution for shRNA-seq screen data, that begins with the raw sequence reads and ends with a ranked lists of candidate shRNAs for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (samples in the columns, hairpins in the rows) with options for allowing mismatches and small shifts in hairpin position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or a generalized linear modelling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from http://www.bioconductor.org.

The miRNAome of the postpartum dairy cow liver in negative energy balance.

BackgroundNegative energy balance (NEB) is an altered metabolic state in high yielding cows that occurs during the first few weeks postpartum when energy demands for lactation and maintenance exceed the energy supply from dietary intake. NEB can, in turn, lead to metabolic disorders and to reduced fertility. Alterations in the expression of more than 700 hepatic genes have previously been reported in a study of NEB in postpartum dairy cows. miRNAs (microRNA) are known to mediate many alterations in gene expression post transcriptionally. To study the hepatic miRNA content of postpartum dairy cows, including their overall abundance and differential expression, in mild NEB (MNEB) and severe NEB (SNEB), short read RNA sequencing was carried out. To identify putative targets of differentially expressed miRNAs among differentially expressed hepatic genes reported previously in dairy cows in SNEB computational target identification was employed.ResultsOur results indicate that the dairy cow liver expresses 53 miRNAs at a lower threshold of 10 reads per million. Of these, 10 miRNAs accounted for greater than 95% of the miRNAome (miRNA content). Of the highly expressed miRNAs, miR-122 constitutes 75% followed by miR-192 and miR-3596. Five out of thirteen let-7 miRNA family members are also among the highly expressed miRNAs. miR-143, down-regulated in SNEB, was found to have 4 putative up-regulated gene targets associated with SNEB including LRP2 (low density lipoprotein receptor-related protein 2), involved in lipid metabolism and up-regulated in SNEB.ConclusionsThis is the first liver miRNA-seq profiling study of moderate yielding dairy cows in the early postpartum period. Tissue specific miR-122 and liver enriched miR-192 are two of the most abundant miRNAs in the postpartum dairy cow liver. miR-143 is significantly down-regulated in SNEB and putative targets of miRNA-143 which are up-regulated in SNEB, include a gene involved in lipid metabolism.

Short RNA indicator sequences are not completely degraded by autoclaving

Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

Preclinical modelling of childhood acute lymphoblastic leukaemia

BackgroundGreatly improved outcomes for children with leukaemia have come at the cost of intensive and toxic treatment regimens. New precision drugs are needed and must be validated in the most representative preclinical models before they can be assessed in clinical trials for children. We have developed an orthotopic patient-derived xenograft model of acute lymphoblastic leukaemia (ALL) that allows real time in-vivo tracking of disease engraftment, candidate gene analysis, and therapeutic validation. MethodsPatient-derived leukaemic blasts were transduced with a lentivector expressing enhanced green fluorescent protein (in-vitro analysis) and firefly luciferase (in-vivo tracking). Transduced cells were injected intrafemorally into highly immunocompromised NOD/LtSz-scid IL-2Rγ-/- (NSG) mice. Stable expression of the lentivector allowed bioluminescent tracking of disease progression over serial transplantations. Modification of the lentivector to include short hairpin RNA sequences has allowed simultaneous in-vivo tracking and gene knockdown. Furthermore, this model has been applied to the validation of a BCL2 inhibitor, ABT-737, as a novel targeted therapy in BCR-ABL positive ALL. FindingsKnockdown of the fusion oncogene MLL/AF4 impeded engraftment of the infant ALL cell line SEM. Treatment of mice with ABT-737 over 6 weeks substantially impaired leukaemia growth and prolonged the median survival from 151 to 190 days (p=0·0004). InterpretationIn this study, we have developed a lentiviral approach to the in-vivo preclinical validation of gene function, coupled with subsequent analysis of novel therapies. It combines use of patient-derived material with a suitable host microenvironment to minimise the risk of false-positive identification of new therapies. This approach minimises the risk to children enrolled into early-phase clinical studies and will accelerate development of effective treatments. FundingUK Medical Research Council, Cancer Research UK grant (C27943/A12788). The IVIS spectrum was funded by Welcome Trust grant 087961.

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule-triggered, dose-dependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet- and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses.

Paxillin is an intrinsic negative regulator of platelet activation in mice

BackgroundPaxillin is a LIM domain protein localized at integrin-mediated focal adhesions. Although paxillin is thought to modulate the functions of integrins, little is known about the contribution of paxillin to signaling pathways in platelets. Here, we studied the role of paxillin in platelet activation in vitro and in vivo.Methods and resultsWe generated paxillin knockdown (Pxn-KD) platelets in mice by transplanting bone marrow cells transduced with a lentiviral vector carrying a short hairpin RNA sequence, and confirmed that paxillin expression was significantly reduced in platelets derived from the transduced cells. Pxn-KD platelets showed a slight increased in size and augmented integrin αIIbβ3 activation following stimulation of multiple receptors including glycoprotein VI and G protein-coupled receptors. Thromboxane A2 biosynthesis and the release of α-granules and dense granules in response to agonist stimulation were also enhanced in Pxn-KD platelets. However, Pxn-KD did not increase tyrosine phosphorylation or intracellular calcium mobilization. Intravital imaging confirmed that Pxn-KD enhanced thrombus formation in vivo.ConclusionsOur findings suggest that paxillin negatively regulates several common platelet signaling pathways, resulting in the activation of integrin αIIbβ3 and release reactions.

Widespread microRNA dysregulation in multiple system atrophy - disease-related alteration in miR-96.

MicroRNA (miRNA) are short sequences of RNA that function as post-transcriptional regulators by binding to target mRNA transcripts resulting in translational repression. A number of recent studies have identified miRNA as being involved in neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the role of miRNA in multiple system atrophy (MSA), a progressive neurodegenerative disorder characterized by oligodendroglial accumulation of alpha-synuclein remains unexamined. In this context, this study examined miRNA profiles in MSA cases compared with controls and in transgenic (tg) models of MSA compared with non-tg mice. The results demonstrate a widespread dysregulation of miRNA in MSA cases, which is recapitulated in the murine models. The study employed a cross-disease, cross-species approach to identify miRNA that were either specifically dysregulated in MSA or were commonly dysregulated in neurodegenerative conditions such as Alzheimer's disease, dementia with Lewy bodies, progressive supranuclear palsy and corticobasal degeneration or the tg mouse model equivalents of these disorders. Using this approach we identified a number of miRNA that were commonly dysregulated between disorders and those that were disease-specific. Moreover, we identified miR-96 as being up-regulated in MSA. Consistent with the up-regulation of miR-96, mRNA and protein levels of members of the solute carrier protein family SLC1A1 and SLC6A6, miR-96 target genes, were down-regulated in MSA cases and a tg model of MSA. These results suggest that miR-96 dysregulation may play a role in MSA and its target genes may be involved in the pathogenesis of MSA.

Using RNA inverse folding to identify IRES-like structural subdomains

Internal ribosome entry site (IRES) elements govern protein synthesis of mRNAs that bypass cap-dependent translation inhibition under stress conditions. Picornavirus IRES are cis-acting elements, organized in modular domains that recruit the ribosome to internal mRNA sites. The aim of this study was to retrieve short RNA sequences with the capacity to adopt RNA folding patterns conserved with IRES structural subdomains, likely corresponding to RNA modules. We have applied a new program, RNAiFold, an inverse folding algorithm that determines all sequences whose minimum free energy structure is identical to that of the structural domains of interest. Sequences differing by more than 1 nt were clustered. Then, BLASTing one randomly chosen sequence from each cluster of the RNAiFold output, we retrieved viral and cellular sequences among output hits. As a proof of principle, we present the data corresponding to a coding region of Drosophila melanogaster TAF6, a transcription factor-associated protein that contains a structural motif within its coding region potentially folding into an IRES-like subdomain. This RNA region shows a biased codon usage, as predicted from structural constraints at the RNA level, it harbors conserved IRES structural motifs in loops, and interestingly, it has the capacity to confer internal initiation of translation in tissue culture cells.

Single-target RNA interference for the blockade of multiple interacting proinflammatory and profibrotic pathways in cardiac fibroblasts

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in “individual” human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to “individual” human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.

Targeted suppression of glutaryl-CoA dehydrogenase by lentivirus-mediated SHRNA and excessive intake of lysine induce the apoptosis of rat striatal neurons

Methods Four short hairpin RNA (shRNA) sequences targeting the GCDH gene (NM_001108896) were designed to construct four recombinant lentiviral vectors. The effectiveness of gene silencing in rat striatal neurons was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting techniques. GCDH deficiency neurons (GCDH neurons), neurons transfected with negative control virus (NC neurons) and not intervention neurons (C neurons) were all incubated with lysine for 24h in concentrations of 0mmol/L, 5mmol/L, 10mmol/L respectively. The viability was measured with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT). The apoptosis of the neurons were detected by Hoechst33342 and PI. Tetramethylrhodamine methyl ester (TMRM) was used to determine the change of mitochondrial membrane potential. The expression of caspase-3, 8, 9, Bax and Bcl-2 were examined by RT-PCR and Western blotting. Results The efficiency of gene silencing of lentivirus-mediated shRNA4 was up to 60%, compared with the parental and control groups. The viability of C neurons, together with mitochondrial membrane potential and expression of caspase-3, 8, 9, Bax/Bcl-2 was not influenced by lysine, even when the concentration was 10mmol/L. The viability of NC neurons was significantly higher than GCDH neurons, when with 5mmol/L lysine interference. When without lysine, there is no difference between the two. Moreover, 5mmol/L lysine could induce GCDH neurons apoptosis, and 10mmmol/L lysine could induce NC neurons apoptosis. In these apoptotic neurons, the mitochondrial membrane potential decrased, the expressions of caspase-3, 8, 9, Bax all increased significantly and Bcl-2 expression decreased, compared to normal cells.

MicroRNA detection in feces, sputum, pleural effusion and urine: Novel tools for cancer screening (Review)

microRNAs (miRNAs) are short non-coding RNA sequences that play important roles in the regulation of gene expression. They have significant regulatory functions in basic cellular processes, including differentiation, proliferation and apoptosis. miRNAs are differentially expressed in tumors, compared with normal tissues. Importantly, miRNAs are also stable and abundantly present in body fluids and feces. The high reproducibility, sensitivity and specificity of miRNAs in body fluids and feces enable miRNAs to be used as potential molecular markers for cancer screening. An increasingly large number of research studies have reported the role of miRNAs in this field. In the present review, we focused mainly on the application of detecting miRNAs in stool, sputum, pleural effusion and urine, to detect colon, lung and urological cancers, highlighting the role of miRNAs in early diagnosis and prognosis.

Novel Biomarkers Assessing Endothelial Dysfunction: Role of microRNAs

Endothelial dysfunction reflected by reduced nitric oxide availability is nowadays considered as a causative factor of atherosclerosis. A variety of biomarkers has been used as indicators of endothelial dysfunction in cardiovascular disease. Discovered just over a decade ago, microRNAs have evoked a great deal of interest, due to their importance for many aspects of homeostasis and disease. miRNAs comprise a novel class of endogenous, single-stranded, short RNA sequences able to regulate gene expression by binding to complementary sequences on mRNAs According to a growing body of evidence, they have been implicated in the regulation of several human physiological processes. They have been shown to participate in cardiovascular disease pathogenesis including atherosclerosis and endothelial dysfunction and this may have important clinical implications.

MicroRNA biomarkers in glioblastoma

Recent research suggests that deregulation of microRNAs (miRNAs) is involved in initiation and progression of many cancers, including gliomas and that miRNAs hold great potential as future diagnostic and therapeutic tools in cancer. MiRNAs are a class of short non-coding RNA sequences (18-24 nucleotides), which base-pair to target messenger RNA (mRNA) and thereby cause translational repression or mRNA degradation based on the level of complementarity between strands. Profiling miRNAs in clinical glioblastoma samples has shown aberrant expression of numerous miRNAs when compared to normal brain tissues. Understanding these alterations is key to developing new biomarkers and intelligent treatment strategies. This review presents an overview of current knowledge about miRNA alterations in glioblastoma while focusing on the clinical future of miRNAs as biomarkers and discussing the strengths and weaknesses of various methods used in evaluating their expression.

Direct evidence for RNA–RNA interactions at the 3′ end of the Hepatitis C virus genome using surface plasmon resonance

Surface plasmon resonance was used to investigate two previously described interactions analyzed by reverse genetics and complementation mutation experiments, involving 5BSL3.2, a stem-loop located in the NS5B coding region of HCV. 5BSL3.2 was immobilized on a sensor chip by streptavidin-biotin coupling, and its interaction either with the SL2 stem-loop of the 3' end or with an upstream sequence centered on nucleotide 9110 (referred to as Seq9110) was monitored in real-time. In contrast with previous results obtained by NMR assays with the same short RNA sequences that we used or SHAPE analysis with longer RNAs, we demonstrate that recognition between 5BSL3.2 and SL2 can occur in solution through a kissing-loop interaction. We show that recognition between Seq9110 and the internal loop of 5BSL3.2 does not prevent binding of SL2 on the apical loop of 5BSL3.2 and does not influence the rate constants of the SL2-5BSL3.2 complex. Therefore, the two binding sites of 5BSL3.2, the apical and internal loops, are structurally independent and both interactions can coexist. We finally show that the stem-loop SL2 is a highly dynamic RNA motif that fluctuates between at least two conformations: One is able to hybridize with 5BSL3.2 through loop-loop interaction, and the other one is capable of self-associating in the absence of protein, reinforcing the hypothesis of SL2 being a dimerization sequence. This result suggests also that the conformational dynamics of SL2 could play a crucial role for controlling the destiny of the genomic RNA.

Abstract A34: Artificial multi-target microRNAs: A new RNA interference approach to enable simultaneous suppression of multiple genes

Abstract RNA interference (RNAi) has become a powerful tool for the suppression of gene expression and the identification of therapeutic targets. RNAi screens have revealed synthetic lethal cancer vulnerabilities, and progress has been made toward enabling therapeutic delivery of short interfering RNAs (siRNAs) in vivo. However, current RNAi libraries are designed to detect synthetic lethal interactions between individual genes and an oncogenic lesion. They are less effective for detecting more subtle interactions in which a synthetic lethal phenotype requires the combined inhibition of several genes in addition to the sensitizing mutation. Identifying such interactions, where multiple genes cooperate with an oncogenic lesion, may open up new avenues for therapeutic intervention, but their discovery remains difficult with current approaches. We therefore sought to develop a new RNAi system in which a single short RNA sequence can suppress multiple genes simultaneously. As the basis for our design, we mimicked the activity of endogenous microRNAs (miRNAs), and refer to our system as “artificial miRNAs.” In contrast to traditional siRNAs and short hairpin RNAs (shRNAs) that are perfectly complementary to their target, miRNAs utilize partial complementarity to direct the repression of multiple genes at the same time. By screening a library of artificial miRNAs, we expect to reveal a novel class of synthetic lethal interactions that can be exploited for cancer therapy. Moreover, in vivo delivery of artificial miRNAs may enable combined inhibition of multiple therapeutic targets that cannot currently be targeted by small molecules. We developed an algorithm to generate artificial miRNA sequences targeting a given set of genes of interest. Based on the mechanisms of endogenous miRNA targeting, our algorithm designs artificial miRNAs that feature perfect complementarity between the miRNA 5'-end “seed region” and each of the target sequences as well as partial complementarity between the miRNA 3' end and each target. To test the hypothesis that miRNAs can be rationally designed for the simultaneous knockdown of multiple genes, we designed and synthesized 234 artificial miRNAs to target two metabolic genes implicated in glioblastoma: pyruvate carboxylase (PC) and glutaminase (GLS). In addition, we generated 74 non-targeting control miRNAs. Using luciferase reporter assays, we observed that PC/GLS-targeted artificial miRNAs were significantly more effective at suppressing both genes than non-targeting controls. We then confirmed by immunoblotting that 13 artificial miRNAs knockdown endogenous PC and GLS simultaneously. Thus, we demonstrated that artificial miRNAs are effective for multi-gene RNAi. We also found that artificial miRNA activity was predicted by the miRNA:target binding energy and the degree of complementarity at the 3' end of the miRNA. Therefore, we are refining the design algorithm to generate artificial miRNAs with enhanced activity. Taken together, our results validate the artificial miRNA system as an effective RNAi-based approach for multiple gene suppression. We are currently developing a library of ~16,000 artificial miRNAs that will permit functional screens to identify combinatorial synthetic lethal interactions. We expect that multi-target artificial miRNAs will provide an important tool for the identification of new therapeutic targets. Citation Format: Jason D. Arroyo, Emily N. Gallichotte, Muneesh Tewari. Artificial multi-target microRNAs: A new RNA interference approach to enable simultaneous suppression of multiple genes. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A34.

Type II phosphatidylinositol 4-kinase β is an integral signaling component of early T cell activation mechanisms

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase β in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase β with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase β in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase β with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase β is a key component in early T cell activation signaling cascades.

Keratin 8 and 18 Loss in Epithelial Cancer Cells Increases Collective Cell Migration and Cisplatin Sensitivity through Claudin1 Up-regulation

Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Physical model of the immune response of bacteria against bacteriophage through the adaptive CRISPR-Cas immune system

Bacteria and archaea have evolved an adaptive, heritable immune system that recognizes and protects against viruses or plasmids. This system, known as the CRISPR-Cas system, allows the host to recognize and incorporate short foreign DNA or RNA sequences, called ‘spacers’ into its CRISPR system. Spacers in the CRISPR system provide a record of the history of bacteria and phage coevolution. We use a physical model to study the dynamics of this coevolution as it evolves stochastically over time. We focus on the impact of mutation and recombination on bacteria and phage evolution and evasion. We discuss the effect of different spacer deletion mechanisms on the coevolutionary dynamics. We make predictions about bacteria and phage population growth, spacer diversity within the CRISPR locus, and spacer protection against the phage population.

The 106b∼25 microRNA cluster is essential for neovascularization after hindlimb ischaemia in mice

MicroRNAs (miRNAs, miR) are endogenous short RNA sequences that regulate a wide range of physiological and pathophysiological processes. Several miRNAs control the formation of new blood vessels either by increasing or by inhibiting angiogenesis. Here, we investigated the possible role of the miR-106b∼25 cluster in postnatal neovascularization and in regulation of the angiogenic properties of adult bone marrow-derived stromal cells. To study the effect of miR-106b∼25 deletion on neovascularization, we used a miR-106b∼25 knockout (KO) mouse model. After inducing hindlimb ischaemia, we showed that vascularization in ischaemic mice devoid of miR-106b∼25 is impaired, as evident from the reduced blood flow on laser Doppler perfusion imaging. The miR-106b∼25 cluster was also shown here to be an essential player in the proper functioning of bone marrow-derived stromal cells through its regulation of apoptosis, matrigel tube formation capacity, cytokine secretion, and expression of the stem-cell marker Sca-1. In addition, we showed that capillary sprouting from miR-106b∼25 KO aortic rings is diminished. These results show that the miR-106b∼25 cluster regulates post-ischaemic neovascularization in mice, and that it does so in part by regulating the function of angiogenic bone marrow-derived stromal cells and of endothelial cells.

Sensing Organic Molecules by Charge Transfer through Aptamer-Target Complexes: Theory and Simulation

Aptamers, i.e., short sequences of RNA and single-stranded DNA, are capable of specificilly binding objects ranging from small molecules over proteins to entire cells. Here, we focus on the structure, stability, dynamics, and electronic properties of oligonucleotides that interact with aromatic or heterocyclic targets. Large-scale molecular dynamics simulations indicate that aromatic rings such as dyes, metabolites, or alkaloides form stable adducts with their oligonucleotide host molecules at least on the simulation time scale. From molecular dynamics snapshots, the energy parameters relevant to Marcus' theory of charge transfer are computed using a modified Su-Schrieffer-Heeger Hamiltonian, permitting an estimate of the charge transfer rates. In many cases, aptamer binding seriously influences the charge transfer kinetics and the charge carrier mobility within the complex, with conductivities up to the nanoampere range for a single complex. We discuss the conductivity properties with reference to potential applications as biosensors.