Journal of Nucleic Acids最新文献

{"title":"PCR amplification and transcription for site-specific labeling of large RNA molecules by a two-unnatural-base-pair system.","authors":"Michiko Kimoto,&nbsp;Rie Yamashige,&nbsp;Shigeyuki Yokoyama,&nbsp;Ichiro Hirao","doi":"10.1155/2012/230943","DOIUrl":"https://doi.org/10.1155/2012/230943","url":null,"abstract":"<p><p>For the site-specific labeling and modification of RNA by genetic alphabet expansion, we developed a PCR and transcription system using two hydrophobic unnatural base pairs: 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) as a third pair for PCR amplification and Ds and pyrrole-2-carbaldehyde (Pa) for the incorporation of functional components as modified Pa bases into RNA by T7 transcription. To prepare Ds-containing DNA templates with long chains, the Ds-Px pair was utilized in a fusion PCR method, by which we demonstrated the synthesis of 282-bp DNA templates containing Ds at specific positions. Using these Ds-containing DNA templates and a biotin-linked Pa substrate (Biotin-PaTP) as a modified Pa base, 260-mer RNA transcripts containing Biotin-Pa at a specific position were generated by T7 RNA polymerase. This two-unnatural-base-pair system, combining the Ds-Px and Ds-Pa pairs with modified Pa substrates, provides a powerful tool for the site-specific labeling and modification of desired positions in large RNA molecules.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"230943"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/230943","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30760822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes.","authors":"Zhenghui Wang, Ke Zhang, Karen L Wooley, John-Stephen Taylor","doi":"10.1155/2012/962652","DOIUrl":"10.1155/2012/962652","url":null,"abstract":"<p><p>Probes for monitoring mRNA expression in vivo are of great interest for the study of biological and biomedical problems, but progress has been hampered by poor signal to noise and effective means for delivering the probes into live cells. Herein we report a PNA·DNA strand displacement-activated fluorescent probe that can image the expression of iNOS (inducible nitric oxide synthase) mRNA, a marker of inflammation. The probe consists of a fluorescein labeled antisense PNA annealed to a shorter DABCYL(plus)-labeled DNA which quenches the fluorescence, but when the quencher strand is displaced by the target mRNA the fluorescence is restored. DNA was used for the quencher strand to facilitate electrostatic binding of the otherwise netural PNA strand to a cationic shell crosslinked knedel-like (cSCK) nanoparticle which can deliver the PNA·DNA duplex probe into cells with less toxicity and greater efficiency than other transfection agents. RAW 264.7 mouse macrophage cells transfected with the iNOS PNA·DNA probe via the cSCK showed a 16 to 54-fold increase in average fluorescence per cell upon iNOS stimulation. The increase was 4 to 7-fold higher than that for a non-complementary probe, thereby validating the ability of a PNA·DNA strand displacement-activated probe to image mRNA expression in vivo.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"962652"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463960/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30969650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential of peptides as inhibitors and mimotopes: selection of carbohydrate-mimetic peptides from phage display libraries.","authors":"Teruhiko Matsubara","doi":"10.1155/2012/740982","DOIUrl":"https://doi.org/10.1155/2012/740982","url":null,"abstract":"<p><p>Glycoconjugates play various roles in biological processes. In particular, oligosaccharides on the surface of animal cells are involved in virus infection and cell-cell communication. Inhibitors of carbohydrate-protein interactions are potential antiviral drugs. Several anti-influenza drugs such as oseltamivir and zanamivir are derivatives of sialic acid, which inhibits neuraminidase. However, it is very difficult to prepare a diverse range of sugar derivatives by chemical synthesis or by the isolation of natural products. In addition, the pathogenic capsular polysaccharides of bacteria are carbohydrate antigens, for which a safe and efficacious method of vaccination is required. Phage-display technology has been improved to enable the identification of peptides that bind to carbohydrate-binding proteins, such as lectins and antibodies, from a large repertoire of peptide sequences. These peptides are known as \"carbohydrate-mimetic peptides (CMPs)\" because they mimic carbohydrate structures. Compared to carbohydrate derivatives, it is easy to prepare mono- and multivalent peptides and then to modify them to create various derivatives. Such mimetic peptides are available as peptide inhibitors of carbohydrate-protein interactions and peptide mimotopes that are conjugated with adjuvant for vaccination.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"740982"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/740982","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31000344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees.","authors":"Philip H Williams, Rod Eyles, Georg Weiller","doi":"10.1155/2012/652979","DOIUrl":"10.1155/2012/652979","url":null,"abstract":"<p><p>MicroRNAs (miRNAs) are nonprotein coding RNAs between 20 and 22 nucleotides long that attenuate protein production. Different types of sequence data are being investigated for novel miRNAs, including genomic and transcriptomic sequences. A variety of machine learning methods have successfully predicted miRNA precursors, mature miRNAs, and other nonprotein coding sequences. MirTools, mirDeep2, and miRanalyzer require \"read count\" to be included with the input sequences, which restricts their use to deep-sequencing data. Our aim was to train a predictor using a cross-section of different species to accurately predict miRNAs outside the training set. We wanted a system that did not require read-count for prediction and could therefore be applied to short sequences extracted from genomic, EST, or RNA-seq sources. A miRNA-predictive decision-tree model has been developed by supervised machine learning. It only requires that the corresponding genome or transcriptome is available within a sequence window that includes the precursor candidate so that the required sequence features can be collected. Some of the most critical features for training the predictor are the miRNA:miRNA(∗) duplex energy and the number of mismatches in the duplex. We present a cross-species plant miRNA predictor with 84.08% sensitivity and 98.53% specificity based on rigorous testing by leave-one-out validation.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"652979"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503367/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31097705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Lighting Up RNA-Cleaving DNAzymes for Biosensing.","authors":"Kha Tram,&nbsp;Pushpinder Kanda,&nbsp;Yingfu Li","doi":"10.1155/2012/958683","DOIUrl":"https://doi.org/10.1155/2012/958683","url":null,"abstract":"<p><p>The development of the in vitro selection technique has allowed the isolation of functional nucleic acids, including catalytic DNA molecules (DNAzymes), from random-sequence pools. The first-ever catalytic DNA obtained by this technique in 1994 is a DNAzyme that cleaves RNA. Since then, many other RNase-like DNAzymes have been reported from multiple in vitro selection studies. The discovery of various RNase DNAzymes has in turn stimulated the exploration of these enzymatic species for innovative applications in many different areas of research, including therapeutics, biosensing, and DNA nanotechnology. One particular research topic that has received considerable attention for the past decade is the development of RNase DNAzymes into fluorescent reporters for biosensing applications. This paper provides a concise survey of the most significant achievements within this research topic.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"958683"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/958683","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31097706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Superior Silencing by 2',4'-BNA(NC)-Based Short Antisense Oligonucleotides Compared to 2',4'-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors.","authors":"Tsuyoshi Yamamoto,&nbsp;Hidenori Yasuhara,&nbsp;Fumito Wada,&nbsp;Mariko Harada-Shiba,&nbsp;Takeshi Imanishi,&nbsp;Satoshi Obika","doi":"10.1155/2012/707323","DOIUrl":"https://doi.org/10.1155/2012/707323","url":null,"abstract":"<p><p>The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2',4'-BNA(NC) antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2',4'-BNA(NC) was directly compared to its conventional bridged (or locked) nucleic acid (2',4'-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2',4'-BNA(NC)-based antisense oligonucleotides and the target mRNA surpassed those of 2',4'-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2',4'-BNA/LNA-based counterpart, the corresponding 2',4'-BNA(NC)-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2',4'-BNA(NC)-based 20-mer AON exhibited the highest affinity but the worst IC(50) value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2',4'-BNA(NC) may be a better alternative to conventional 2',4'-BNA/LNA, even for \"short\" antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"707323"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/707323","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30969649","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nucleic-Acid-binding chromophores as efficient indicators of aptamer-target interactions.","authors":"Kwabena Sarpong,&nbsp;Bhaskar Datta","doi":"10.1155/2012/247280","DOIUrl":"https://doi.org/10.1155/2012/247280","url":null,"abstract":"<p><p>The binding affinity and specificity of nucleic acid aptamers have made them valuable candidates for use as sensors in diagnostic applications. In particular, chromophore-functionalized aptamers offer a relatively simple format for detection and quantification of target molecules. We describe the use of nucleic-acid-staining reagents as an effective tool for detecting and signaling aptamer-target interactions. Aptamers varying in size and structure and targeting a range of molecules have been used in conjunction with commercially available chromophores to indicate and quantify the presence of cognate targets with high sensitivity and selectivity. Our assay precludes the covalent modification of nucleic acids and relies on the differential fluorescence signal of chromophores when complexed with aptamers with or without their cognate target. We also evaluate factors that are critical for the stability of the complex between the aptamer and chromophore in presence or absence of target molecules. Our results indicate the possibility of controlling those factors to enhance the sensitivity of target detection by the aptamers used in such assays.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"247280"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/247280","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"31000341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alternative splicing and cancer.","authors":"Didier Auboeuf,&nbsp;Maria Carmo-Fonseca,&nbsp;Juan Valcarcel,&nbsp;Giuseppe Biamonti","doi":"10.1155/2012/363809","DOIUrl":"https://doi.org/10.1155/2012/363809","url":null,"abstract":"Alternative splicing of premessenger RNAs is a key step in the gene expression process, which allows the synthesis of different products from the same gene and contributes to increase the complexity of the proteome coded by a limited number of genes. Specialized high-throughput technologies (RNA-Seq, splicing-sensitive microarrays) aiming at analyzing alternative splicing in normal or pathological situations have allowed to make a promising step forward in basic and translational molecular oncology by identifying a variety of cancer-associated splicing variants. However, modification of alternative splicing is among the myriad of alterations present in cancer cells and whether splicing alteration is a cause or a consequence of cancer remains to be elucidated. \u0000 \u0000The main focus of this special issue is to highlight some of the mechanisms involved in splicing alteration in cancer and to present new evidence demonstrating the involvement of alternative splicing alterations in different steps and aspects of cancer initiation and progression. \u0000 \u0000To highlight the applications of large-scale approaches in the search for relevant cancer-associated splicing events, S. Germann and colleagues give an overview of the studies that have been carried out so far using such strategies. This has allowed to identify sets of functionally related genes whose expression is altered at the splicing level in cancer cells and to characterize some of the factors which control specific splicing programs that are deregulated in tumors. \u0000 \u0000The other reviews give a series of specific examples of cancer-associated splicing variants. S. Druillennec and colleagues address how alternative splicing modifies the physiological and pathological functions of a variety of protein kinases. Taking several examples of membrane-associated or cytosolic kinases, they explain more particularly how the oncogenic properties of this important class of factors between specific splicing isoforms. \u0000 \u0000More specifically, K. Holzmann and colleagues summarize the various splicing alterations that affect, in different tumor types, the transcripts encoding the fibroblast growth factor receptors (FGFR) 1–3, at the level of their IgIII loop. Splicing-induced variations in this domain, which occur naturally during embryonic development and are regulated in a tissue-specific manner, directly affect the interactions between the receptors and their ligands and have profound consequences on their activity. In cancer cells, alterations in FGFR2 splicing are involved in the epithelial-mesenchymal transition, an important step in the formation of metastases. \u0000 \u0000In their paper, Hilmi and colleagues focus on the alternative splicing of vascular endothelial growth factor (VEGF), which produces isoforms with opposite functions in the control of angiogenesis, a process involved in the progression and metastasis of several cancers. They discuss the emerging possibility of targeting angiogenesis more accurately by modulat","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"363809"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/363809","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30680836","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MsDpo4-a DinB Homolog from Mycobacterium smegmatis-Is an Error-Prone DNA Polymerase That Can Promote G:T and T:G Mismatches.","authors":"Amit Sharma,&nbsp;Deepak T Nair","doi":"10.1155/2012/285481","DOIUrl":"https://doi.org/10.1155/2012/285481","url":null,"abstract":"<p><p>Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":" ","pages":"285481"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/285481","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40174121","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"VEGF spliced variants: possible role of anti-angiogenesis therapy.","authors":"Caroline Hilmi,&nbsp;Mélanie Guyot,&nbsp;Gilles Pagès","doi":"10.1155/2012/162692","DOIUrl":"https://doi.org/10.1155/2012/162692","url":null,"abstract":"<p><p>Angiogenesis has been targeted in retinopathies, psoriasis, and a variety of cancers (colon, breast, lung, and kidney). Among these tumour types, clear cell renal cell carcinomas (RCCs) are the most vascularized tumours due to mutations of the von Hippel Lindau gene resulting in HIF-1 alpha stabilisation and overexpression of Vascular Endothelial Growth Factor (VEGF). Surgical nephrectomy remains the most efficient curative treatment for patients with noninvasive disease, while VEGF targeting has resulted in varying degrees of success for treating metastatic disease. VEGF pre-mRNA undergoes alternative splicing generating pro-angiogenic isoforms. However, the recent identification of novel splice variants of VEGF with anti-angiogenic properties has provided some insight for the lack of current treatment efficacy. Here we discuss an explanation for the relapse to anti-angiogenesis treatment as being due to either an initial or acquired resistance to the therapy. We also discuss targeting angiogenesis via SR (serine/arginine-rich) proteins implicated in VEGF splicing.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":"2012 ","pages":"162692"},"PeriodicalIF":2.3,"publicationDate":"2012-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1155/2012/162692","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"30220978","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}