Journal of Nucleic Acids http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins Tue, 15 Apr 2014 14:18:04 +0000 http://www.hindawi.com/journals/jna/2014/214929/ Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells. Sreerupa Challa, Saul Tzipori, and Abhineet Sheoran Copyright © 2014 Sreerupa Challa et al. All rights reserved. Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit Sun, 29 Dec 2013 11:51:41 +0000 http://www.hindawi.com/journals/jna/2013/763945/ The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific. Manassés Daniel da Silva, Roberta Lane de Oliveira Silva, José Ribamar Costa Ferreira Neto, Ana Carolina Ribeiro Guimarães, Daniela Truffi Veiga, Sabrina Moutinho Chabregas, William Lee Burnquist, Günter Kahl, Ana Maria Benko-Iseppon, and Ederson Akio Kido Copyright © 2013 Manassés Daniel da Silva et al. All rights reserved. Multipyrene Tandem Probes for Point Mutations Detection in DNA Tue, 24 Dec 2013 08:12:21 +0000 http://www.hindawi.com/journals/jna/2013/860457/ Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. Svetlana A. Kholodar, Darya S. Novopashina, Mariya I. Meschaninova, and Alya G. Venyaminova Copyright © 2013 Svetlana A. Kholodar et al. All rights reserved. Transcriptionally Repressive Chromatin Remodelling and CpG Methylation in the Presence of Expanded CTG-Repeats at the DM1 Locus Mon, 23 Dec 2013 15:28:42 +0000 http://www.hindawi.com/journals/jna/2013/567435/ An expanded CTG-repeat in the 3′ UTR of the DMPK gene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreased DMPK sense and SIX5 transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability. Judith Rixt Brouwer, Aline Huguet, Annie Nicole, Arnold Munnich, and Geneviève Gourdon Copyright © 2013 Judith Rixt Brouwer et al. All rights reserved. Comparative (Computational) Analysis of the DNA Methylation Status of Trinucleotide Repeat Expansion Diseases Mon, 23 Dec 2013 09:51:50 +0000 http://www.hindawi.com/journals/jna/2013/689798/ Previous studies have examined DNA methylation in different trinucleotide repeat diseases. We have combined this data and used a pattern searching algorithm to identify motifs in the DNA surrounding aberrantly methylated CpGs found in the DNA of patients with one of the three trinucleotide repeat (TNR) expansion diseases: fragile X syndrome (FRAXA), myotonic dystrophy type I (DM1), or Friedreich’s ataxia (FRDA). We examined sequences surrounding both the variably methylated (VM) CpGs, which are hypermethylated in patients compared with unaffected controls, and the nonvariably methylated CpGs which remain either always methylated (AM) or never methylated (NM) in both patients and controls. Using the J48 algorithm of WEKA analysis, we identified that two patterns are all that is necessary to classify our three regions CCGG* which is found in VM and not in AM regions and AATT* which distinguished between NM and VM + AM using proportional frequency. Furthermore, comparing our software with MEME software, we have demonstrated that our software identifies more patterns than MEME in these short DNA sequences. Thus, we present evidence that the DNA sequence surrounding CpG can influence its susceptibility to be de novo methylated in a disease state associated with a trinucleotide repeat. Mohammadmersad Ghorbani, Simon J. E. Taylor, Mark A. Pook, and Annette Payne Copyright © 2013 Mohammadmersad Ghorbani et al. All rights reserved. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis Tue, 17 Dec 2013 10:36:09 +0000 http://www.hindawi.com/journals/jna/2013/801505/ Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. John P. Jakupciak, Jeffrey M. Wells, Richard J. Karalus, David R. Pawlowski, Jeffrey S. Lin, and Andrew B. Feldman Copyright © 2013 John P. Jakupciak et al. All rights reserved. Oligonucleotide-Based Therapy for FTD/ALS Caused by the C9orf72 Repeat Expansion: A Perspective Sun, 17 Nov 2013 10:51:58 +0000 http://www.hindawi.com/journals/jna/2013/208245/ Amyotrophic lateral sclerosis (ALS) is a progressive and lethal disease of motor neuron degeneration, leading to paralysis of voluntary muscles and death by respiratory failure within five years of onset. Frontotemporal dementia (FTD) is characterised by degeneration of frontal and temporal lobes, leading to changes in personality, behaviour, and language, culminating in death within 5–10 years. Both of these diseases form a clinical, pathological, and genetic continuum of diseases, and this link has become clearer recently with the discovery of a hexanucleotide repeat expansion in the C9orf72 gene that causes the FTD/ALS spectrum, that is, c9FTD/ALS. Two basic mechanisms have been proposed as being potentially responsible for c9FTD/ALS: loss-of-function of the protein encoded by this gene (associated with aberrant DNA methylation) and gain of function through the formation of RNA foci or protein aggregates. These diseases currently lack any cure or effective treatment. Antisense oligonucleotides (ASOs) are modified nucleic acids that are able to silence targeted mRNAs or perform splice modulation, and the fact that they have proved efficient in repeat expansion diseases including myotonic dystrophy type 1 makes them ideal candidates for c9FTD/ALS therapy. Here, we discuss potential mechanisms and challenges for developing oligonucleotide-based therapy for c9FTD/ALS. Stephanie A. Fernandes, Andrew G. L. Douglas, Miguel A. Varela, Matthew J. A. Wood, and Yoshitsugu Aoki Copyright © 2013 Stephanie A. Fernandes et al. All rights reserved. Immobilization of DNA Aptamers on Polyester Cloth for Antigen Detection by Dot Blot Immunoenzymatic Assay (Aptablot) Wed, 30 Oct 2013 11:20:57 +0000 http://www.hindawi.com/journals/jna/2013/936542/ A simple dot blot immunoenzymatic assay system was developed using polyester cloth coated with an oligo-DNA aptamer to provide a high-affinity macroporous surface for the efficient capture of a model protein analyte (thrombin) in complex sample matrices such as foods. Bound thrombin was detected immunoenzymatically using a peroxidase-linked antithrombin antibody and a chromogenic substrate. A unique feature of this approach, which we have termed “aptablot,” is the facile immobilization of DNA aptamers on the polyester surface by cross-linking with a brief exposure to ultraviolet light, and the simple assay format obviating the need for specialized instruments. The assay principle described herein should be broadly applicable to many situations where analytes must be detected in complex samples, with the main limiting factor being the availability of suitable DNA aptamers. Sally Smiley, Maria DeRosa, and Burton Blais Copyright © 2013 Sally Smiley et al. All rights reserved. Simultaneous Detection of Different MicroRNA Types Using the ZIP-Code Array System Mon, 02 Sep 2013 08:16:18 +0000 http://www.hindawi.com/journals/jna/2013/496425/ MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells. Sonja U. Weishaupt, Steffen Rupp, and Karin Lemuth Copyright © 2013 Sonja U. Weishaupt et al. All rights reserved. R-Loop Formation In Trans at an AGGAG Repeat Mon, 26 Aug 2013 08:34:06 +0000 http://www.hindawi.com/journals/jna/2013/629218/ Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling. Kazuya Toriumi, Takuma Tsukahara, and Ryo Hanai Copyright © 2013 Kazuya Toriumi et al. All rights reserved. Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR Sun, 21 Jul 2013 13:34:28 +0000 http://www.hindawi.com/journals/jna/2013/823730/ Thermus thermophilus MutS, a thermostable mismatch-recognizing protein, is utilized in PCR to suppress nonspecific amplification by preventing synthesis from mismatched primers. T. thermophilus RecA also decreases nonspecific amplification by promoting proper hybridization between the primer and template. We observed that MutS and RecA function under the same reaction conditions and that MutS and RecA do not preclude each other. Furthermore, there were some DNA sequences for which only one of the 2 proteins effectively suppressed nonspecific amplification. The simultaneous use of MutS and RecA is a more attractive error-suppressing technique than the use of either of the 2 proteins alone. Kenji Fukui and Seiki Kuramitsu Copyright © 2013 Kenji Fukui and Seiki Kuramitsu. All rights reserved. Mesenchymal Stem Cell Therapy in Diabetes Mellitus: Progress and Challenges Wed, 15 May 2013 07:57:14 +0000 http://www.hindawi.com/journals/jna/2013/194858/ Advanced type 2 diabetes mellitus is associated with significant morbidity and mortality due to cardiovascular, nervous, and renal complications. Attempts to cure diabetes mellitus using islet transplantation have been successful in providing a source for insulin secreting cells. However, limited donors, graft rejection, the need for continued immune suppression, and exhaustion of the donor cell pool prompted the search for a more sustained source of insulin secreting cells. Stem cell therapy is a promising alternative for islet transplantation in type 2 diabetic patients who fail to control hyperglycemia even with insulin injection. Autologous stem cell transplantation may provide the best outcome for those patients, since autologous cells are readily available and do not entail prolonged hospital stays or sustained immunotoxic therapy. Among autologous adult stem cells, mesenchymal stem cells (MSCs) therapy has been applied with varying degrees of success in both animal models and in clinical trials. This review will focus on the advantages of MSCs over other types of stem cells and the possible mechanisms by which MSCs transplant restores normoglycemia in type 2 diabetic patients. Sources of MSCs including autologous cells from diabetic patients and the use of various differentiation protocols in relation to best transplant outcome will be discussed. Nagwa El-Badri and Mohamed A. Ghoneim Copyright © 2013 Nagwa El-Badri and Mohamed A. Ghoneim. All rights reserved. Thermodynamic and Structural Analysis of DNA Damage Architectures Related to Replication Sun, 28 Apr 2013 10:35:00 +0000 http://www.hindawi.com/journals/jna/2013/867957/ Damaged DNA, generated by the abstraction of one of five hydrogen atoms from the 2′-deoxyribose ring of the nucleic acid, can contain a variety of lesions, some of which compromise physiological processes. Recently, DNA damage, resulting from the formation of a C3′-thymidinyl radical in DNA oligomers, was found to be dependent on nucleic acid structure. Architectures relevant to DNA replication were observed to generate larger amounts of strand-break and 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine formation than that observed for duplex DNA. To understand how this damage can affect the integrity of DNA, the impact of C3′-thymidinyl radical derived lesions on DNA stability and structure was characterized using biophysical methods. DNA architectures evaluated include duplex DNA (dsDNA), single 3′ or 5′-overhangs (OvHgs), and forks. Thermal melting analysis and differential scanning calorimetry measurements indicate that an individual 3′-OvHg is more destabilizing than a 5′-OvHg. The presence of a terminal 3′ or 5′ phosphate decreases the to the same extent, while the effect of the phosphate at the ss-dsDNA junction of OvHgs is dependent on sequence. Additionally, the effect of 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine is found to depend on DNA architecture and proximity to the 3′ end of the damaged strand. Nicholas J. Amato, Christopher N. Mwai, Timothy C. Mueser, and Amanda C. Bryant-Friedrich Copyright © 2013 Nicholas J. Amato et al. All rights reserved. Base Composition Characteristics of Mammalian miRNAs Sun, 24 Feb 2013 10:25:48 +0000 http://www.hindawi.com/journals/jna/2013/951570/ MicroRNAs (miRNAs) are short RNA sequences that repress protein synthesis by either inhibiting the translation of messenger RNA (mRNA) or increasing mRNA degradation. Endogenous miRNAs have been found in various organisms, including animals, plants, and viruses. Mammalian miRNAs are evolutionarily conserved, are scattered throughout chromosomes, and play an important role in the immune response and the onset of cancer. For this study, the author explored the base composition characteristics of miRNA genes from the six mammalian species that contain the largest number of known miRNAs. It was found that mammalian miRNAs are evolutionarily conserved and GU-rich. Interestingly, in the miRNA sequences investigated, A residues are clearly the most frequent occupants of positions 2 and 3 of the 5′ end of miRNAs. Unlike G and U residues that may pair with C/U and A/G, respectively, A residues can only pair with U residues of target mRNAs, which may augment the recognition specificity of the 5′ seed region. Bin Wang Copyright © 2013 Bin Wang. All rights reserved. Artificially Created Nucleic Acids and Peptides/Proteins in Chemical Biology Tue, 15 Jan 2013 10:28:27 +0000 http://www.hindawi.com/journals/jna/2013/219263/ Masayasu Kuwahara, Yingfu Li, Eriks Rozners, and Hiroshi Murakami Copyright © 2013 Masayasu Kuwahara et al. All rights reserved. Using Aptamers for Cancer Biomarker Discovery Tue, 15 Jan 2013 10:07:58 +0000 http://www.hindawi.com/journals/jna/2013/817350/ Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment) SELEX and cell-based SELEX (cell-SELEX). Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics. Yun Min Chang, Michael J. Donovan, and Weihong Tan Copyright © 2013 Yun Min Chang et al. All rights reserved. Expansion of the Genetic Alphabet: Unnatural Nucleobases and Their Applications Mon, 17 Dec 2012 15:27:55 +0000 http://www.hindawi.com/journals/jna/2012/718582/ Subhendu Sekhar Bag, Jennifer M. Heemstra, Yoshio Saito, and David M. Chenoweth Copyright © 2012 Subhendu Sekhar Bag et al. All rights reserved. Lighting Up RNA-Cleaving DNAzymes for Biosensing Thu, 08 Nov 2012 16:14:18 +0000 http://www.hindawi.com/journals/jna/2012/958683/ 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. Kha Tram, Pushpinder Kanda, and Yingfu Li Copyright © 2012 Kha Tram et al. All rights reserved. Plant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees Wed, 07 Nov 2012 11:59:58 +0000 http://www.hindawi.com/journals/jna/2012/652979/ 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. Philip H. Williams, Rod Eyles, and Georg Weiller Copyright © 2012 Philip H. Williams et al. All rights reserved. Specific Roles of MicroRNAs in Their Interactions with Environmental Factors Wed, 31 Oct 2012 11:32:43 +0000 http://www.hindawi.com/journals/jna/2012/978384/ MicroRNAs (miRNAs) have emerged as critical regulators of gene expression by modulating numerous target mRNAs expression at posttranscriptional level. Extensive studies have shown that miRNAs are critical in various important biological processes, including cell growth, proliferation, differentiation, development, and apoptosis. In terms of their importance, miRNA dysfunction has been associated with a broad range of diseases. Increased number of studies have shown that miRNAs can functionally interact with a wide spectrum of environmental factors (EFs) including drugs, industrial materials, virus and bacterial pathogens, cigarette smoking, alcohol, nutrition, sleep, exercise, stress, and radiation. More importantly, the interactions between miRNAs and EFs have been shown to play critical roles in determining abnormal phenotypes and diseases. In this paper, we propose an outline of the current knowledge about specific roles of miRNAs in their interactions with various EFs and analyze the literatures detailing miRNAs-EFs interactions in the context of various of diseases. Juan Wang and Qinghua Cui Copyright © 2012 Juan Wang and Qinghua Cui. All rights reserved. Dioxaphosphorinane-Constrained Nucleic Acid Dinucleotides as Tools for Structural Tuning of Nucleic Acids Wed, 24 Oct 2012 16:24:24 +0000 http://www.hindawi.com/journals/jna/2012/215876/ We describe a rational approach devoted to modulate the sugar-phosphate backbone geometry of nucleic acids. Constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. The so-called dioxaphosphorinane rings were introduced at key positions along the sugar-phosphate backbone allowing the control of the six-torsion angles to ζ defining the polymer structure. The syntheses of all the members of the D-CNA family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of ,-D-CNA exhibiting wether B-type canonical values or not. Dan-Andrei Catana, Brice-Loïc Renard, Marie Maturano, Corinne Payrastre, Nathalie Tarrat, and Jean-Marc Escudier Copyright © 2012 Dan-Andrei Catana et al. All rights reserved. Challenges and Opportunities for Small Molecule Aptamer Development Wed, 24 Oct 2012 15:54:35 +0000 http://www.hindawi.com/journals/jna/2012/748913/ Aptamers are single-stranded oligonucleotides that bind to targets with high affinity and selectivity. Their use as molecular recognition elements has emerged as a viable approach for biosensing, diagnostics, and therapeutics. Despite this potential, relatively few aptamers exist that bind to small molecules. Small molecules are important targets for investigation due to their diverse biological functions as well as their clinical and commercial uses. Novel, effective molecular recognition probes for these compounds are therefore of great interest. This paper will highlight the technical challenges of aptamer development for small molecule targets, as well as the opportunities that exist for their application in biosensing and chemical biology. Maureen McKeague and Maria C. DeRosa Copyright © 2012 Maureen McKeague and Maria C. DeRosa. All rights reserved. Genetically Encoded Libraries of Nonstandard Peptides Sun, 14 Oct 2012 15:08:00 +0000 http://www.hindawi.com/journals/jna/2012/713510/ The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides. Takashi Kawakami and Hiroshi Murakami Copyright © 2012 Takashi Kawakami and Hiroshi Murakami. All rights reserved. Nucleic-Acid-Binding Chromophores as Efficient Indicators of Aptamer-Target Interactions Wed, 10 Oct 2012 15:12:46 +0000 http://www.hindawi.com/journals/jna/2012/247280/ 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. Kwabena Sarpong and Bhaskar Datta Copyright © 2012 Kwabena Sarpong and Bhaskar Datta. All rights reserved. Potential of Peptides as Inhibitors and Mimotopes: Selection of Carbohydrate-Mimetic Peptides from Phage Display Libraries Wed, 10 Oct 2012 11:20:04 +0000 http://www.hindawi.com/journals/jna/2012/740982/ 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. Teruhiko Matsubara Copyright © 2012 Teruhiko Matsubara. All rights reserved. Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries Mon, 08 Oct 2012 08:05:45 +0000 http://www.hindawi.com/journals/jna/2012/156482/ Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described. Yuuya Kasahara and Masayasu Kuwahara Copyright © 2012 Yuuya Kasahara and Masayasu Kuwahara. All rights reserved. Functional Annotation of Small Noncoding RNAs Target Genes Provides Evidence for a Deregulated Ubiquitin-Proteasome Pathway in Spinocerebellar Ataxia Type 1 Wed, 03 Oct 2012 18:38:51 +0000 http://www.hindawi.com/journals/jna/2012/672536/ Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by the expansion of CAG repeats in the ataxin 1 (ATXN1) gene. In affected cerebellar neurons of patients, mutant ATXN1 accumulates in ubiquitin-positive nuclear inclusions, indicating that protein misfolding is involved in SCA1 pathogenesis. In this study, we functionally annotated the target genes of the small noncoding RNAs (ncRNAs) that were selectively activated in the affected brain compartments. The primary targets of these RNAs, which exhibited a significant enrichment in the cerebellum and cortex of SCA1 patients, were members of the ubiquitin-proteasome system. Thus, we identified and functionally annotated a plausible regulatory pathway that may serve as a potential target to modulate the outcome of neurodegenerative diseases. Stephan Persengiev, Ivanela Kondova, and Ronald E. Bontrop Copyright © 2012 Stephan Persengiev et al. All rights reserved. UvrD Participation in Nucleotide Excision Repair Is Required for the Recovery of DNA Synthesis following UV-Induced Damage in Escherichia coli Thu, 27 Sep 2012 10:32:32 +0000 http://www.hindawi.com/journals/jna/2012/271453/ UvrD is a DNA helicase that participates in nucleotide excision repair and several replication-associated processes, including methyl-directed mismatch repair and recombination. UvrD is capable of displacing oligonucleotides from synthetic forked DNA structures in vitro and is essential for viability in the absence of Rep, a helicase associated with processing replication forks. These observations have led others to propose that UvrD may promote fork regression and facilitate resetting of the replication fork following arrest. However, the molecular activity of UvrD at replication forks in vivo has not been directly examined. In this study, we characterized the role UvrD has in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that UvrD is required for DNA synthesis to recover. However, in the absence of UvrD, the displacement and partial degradation of the nascent DNA at the arrested fork occur normally. In addition, damage-induced replication intermediates persist and accumulate in uvrD mutants in a manner that is similar to that observed in other nucleotide excision repair mutants. These data indicate that, following arrest by DNA damage, UvrD is not required to catalyze fork regression in vivo and suggest that the failure of uvrD mutants to restore DNA synthesis following UV-induced arrest relates to its role in nucleotide excision repair. Kelley N. Newton, Charmain T. Courcelle, and Justin Courcelle Copyright © 2012 Kelley N. Newton et al. All rights reserved. Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes Wed, 26 Sep 2012 10:28:10 +0000 http://www.hindawi.com/journals/jna/2012/962652/ 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 -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. Zhenghui Wang, Ke Zhang, Karen L. Wooley, and John-Stephen Taylor Copyright © 2012 Zhenghui Wang et al. All rights reserved. Superior Silencing by 2′,4′-BNANC-Based Short Antisense Oligonucleotides Compared to 2′,4′-BNA/LNA-Based Apolipoprotein B Antisense Inhibitors Wed, 26 Sep 2012 09:53:59 +0000 http://www.hindawi.com/journals/jna/2012/707323/ The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, ,- antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. ,- was directly compared to its conventional bridged (or locked) nucleic acid (,-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between ,--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 ,-BNA/LNA-based counterpart, the corresponding ,--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′-BNANC-based 20-mer AON exhibited the highest affinity but the worst 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 ,- may be a better alternative to conventional ,-BNA/LNA, even for “short” antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production. Tsuyoshi Yamamoto, Hidenori Yasuhara, Fumito Wada, Mariko Harada-Shiba, Takeshi Imanishi, and Satoshi Obika Copyright © 2012 Tsuyoshi Yamamoto et al. All rights reserved.