Journal of Nucleic Acids最新文献

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.

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.

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.

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.

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.

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.

The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA.

Alternative splicing of the IgIII loop of fibroblast growth factor receptors (FGFRs) 1-3 produces b- and c-variants of the receptors with distinctly different biological impact based on their distinct ligand-binding spectrum. Tissue-specific expression of these splice variants regulates interactions in embryonic development, tissue maintenance and repair, and cancer. Alterations in FGFR2 splicing are involved in epithelial mesenchymal transition that produces invasive, metastatic features during tumor progression. Recent research has elucidated regulatory factors that determine the splice choice both on the level of exogenous signaling events and on the RNA-protein interaction level. Moreover, methodology has been developed that will enable the in depth analysis of splicing events during tumorigenesis and provide further insight on the role of FGFR 1-3 IIIb and IIIc in the pathophysiology of various malignancies. This paper aims to summarize expression patterns in various tumor types and outlines possibilities for further analysis and application.

TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.