Use Of Nucleic Acids Research Articles

Engineered cells as biosensing systems in biomedical analysis

Over the past two decades there have been great advances in biotechnology, including use of nucleic acids, proteins, and whole cells to develop a variety of molecular analytical tools for diagnostic, screening, and pharmaceutical applications. Through manipulation of bacterial plasmids and genomes, bacterial whole-cell sensing systems have been engineered that can serve as novel methods for analyte detection and characterization, and as more efficient and cost-effective alternatives to traditional analytical techniques. Bacterial cell-based sensing systems are typically sensitive, specific and selective, rapid, easy to use, low-cost, and amenable to multiplexing, high-throughput, and miniaturization for incorporation into portable devices. This critical review is intended to provide an overview of available bacterial whole-cell sensing systems for assessment of a variety of clinically relevant analytes. Specifically, we examine whole-cell sensing systems for detection of bacterial quorum sensing molecules, organic and inorganic toxic compounds, and drugs, and for screening of antibacterial compounds for identification of their mechanisms of action. Methods used in the design and development of whole-cell sensing systems are also reviewed.

Unexpected Sophistication and Complexity of Chemomechanical Control Linkages in Nucleic Acid Motor

Ring-shaped, oligomeric ATPases are essential for a variety of cellular processes ranging from protein and nucleic acid metabolism to organelle transport. A subset of these motor proteins, the hexameric helicases, couple the binding and hydrolysis of ATP to the physical manipulation of nucleic acids to support essential cellular processes such as gene regulation, DNA replication, and DNA repair. How nucleotide turnover is coordinated between six independent motor subunits to generate helicase movement has been a long-standing question in the field. The topological problem of how extended nucleic substrates are loaded into the central pore of a closed hexameric toroid is similarly not well-understood.Using the 300 kDa E. coli Rho protein as a model hexameric RNA/DNA helicase, we have examined these issues through a combined structural and biochemical approach. Models derived from high-resolution crystallographic and solution X-ray scattering data explain in molecular detail how the Rho ring opens and closes in response to substrate binding, how the ATP cycle is coordinated with RNA movement through the motor interior, and why certain helicase families translocate with a preferred directional polarity. We find an unexpectedly rich array of physical connectors that sense the RNA binding status of individual subunits in the motor, and that appear to couple these states to the timing of ATP hydrolysis in a manner that is responsive to the base sequence of the substrate. These studies highlight the physical complexity of hexameric helicases, and suggest that related motor proteins utilize similarly intricate chemomechanical linkage mechanisms.

Translational Pathology of Early Cancer

In the last decade, therapy for specific types of cancer has evolved from a standard treatment for each type of cancer to variable forms therapy, the choice of which is dependent upon the analysis and progression of the individual cancer. Characteristics of the tumor, from its clinical manifestation and stage to the cancer’s molecular and pathologic features are beginning to play a major role in the selection of appropriate care of patients with malignant tumors. The visual pathologic features of tumors have always been the critical parameters in determining tumor type; however, over the last several decades molecular analysis of tumors has played an increasing role in the diagnostic pathology of tumors; thus, “molecular pathology” has gradually emerged as a discipline in pathology which exploits the use of nucleic acid based techniques such as DNA sequencing, fluorescent in-situ hybridization, real time quantitative polymerase chain reaction, nucleic acid microarrays for specialized studies, proteomic based techniques and antibody-based profiling of tissues and cells. Molecular pathology shares some aspects of practice with both anatomic and clinical pathology, and is sometimes considered a crossover discipline. Applications are found in studies of both hereditary and acquired disorders including inheritable genetic diseases, malignant tumors, hematological diseases (leukemias and lymphomas), and infectious diseases. For example, DNA testing is the most common method for identity testing with specific applications such as monitoringbonemarrow engraftment, paternity testing, and forensic identification. The transformation of normal cells to malignant cells involves multiple genetic and epigenetic changes that are currently being studied and defined. Many of these changes have already been defined; however, as these studies proceed, we should take advantage of any identified molecular changes to aid in the early detection of cancer and the corresponding preinvasive neoplastic lesions. Small cancers may be successfully treated and even cured, and treating preinvasive neoplastic lesions may prevent the subsequent development of invasive cancer. Molecular detection offers many advantages in the detection of small tumors or preinvasive neoplastic changes. First, molecularmethodswill permit the identification of very small tumors in their earliest stages of development. These tumors can be treated successfully and cost effectively. Current methods of early detection, such as mammography, endoscopic examination with fine needle aspiration or other techniques of imaging, only detect cancers that have grown to a certain, albeit small size. In many cases, however, aggressive subtypes of small tumors may have already invaded blood and lymphatic vessels or have spread to regional lymph nodes. Secondly, molecular methods will eventually detect field changes especially those that affect broad epithelial areas. Because cancer often arises in fields primed for neoplastic development, in the future, treatments will be directed at field changes rather than at individual lesions. The ultimate objective of early detection, therefore, is the diagnosis of tumor initiation and the use of preventive methods to reverse field effects. A primary goal of using molecular methods should be the ability to separate those preinvasive neoplastic lesions that are destined to progress from those that will not, avoiding the over treatment of harmless lesions. In diagnostic pathology, some lesions are diagnosed as ‘borderline’, ‘atypical’ or dysplastic or of indeterminate malignant potential; such terms reflect

Kingella kingae: An Emerging Pathogen in Young Children

Kingella kingae is being recognized increasingly as a common etiology of pediatric osteoarticular infections, bacteremia, and endocarditis, which reflects improved culture methods and use of nucleic acid-amplification techniques in clinical microbiology laboratories. K kingae colonizes the posterior pharynx of young children and is transmitted from child to child through close personal contact. Day care attendance increases the risk for colonization and transmission, and clusters of K kingae infections among day care center attendees have been reported. Key virulence factors in K kingae include type IV pili and a potent RTX toxin. In previously healthy children, >95% of K kingae infections are diagnosed between the ages of 6 and 48 months. Among children with underlying medical conditions, K kingae disease may occur at older ages as well. The clinical presentation of K kingae disease is often subtle and may be associated with normal levels of acute-phase reactants, which underscores the importance of a high index of suspicion. K kingae is usually susceptible to ß-lactam antibiotics, and infections typically respond well to medical treatment, with the exception of cases of endocarditis.

Binding and purification of plasmid DNA using multi-layered carbon nanotubes

We propose a new method for the separation of nucleic acids using multi-layered carbon nanotubes (CNTs) as an adsorbent. According to agarose gel electrophoresis, oxidized water-stable CNTs adsorb certain forms of nucleic acids, such as high molecular weight RNA, chromosomal DNA, linear and denatured forms of plasmid DNA. However, CNTs do not adsorb supercoiled form of plasmid DNA. Nucleic acids bound to CNTs can be readily removed by centrifugation whereas supercoiled plasmid DNA remains in solution. Upon the addition of divalent metal ions supercoiled plasmid DNA forms relatively stable complexes with CNTs due to chelation. Thus, new details about association of nucleic acids with CNTs were revealed and stoichiometry of the complexes was estimated. Our results can be used for fine purification of supercoiled plasmid DNA for gene therapy applications as well as manipulation of nucleic acids for biosensor design.

Survey on indirect optical manipulation of cells, nucleic acids, and motor proteins

Optical tweezers have emerged as a promising technique for manipulating biological objects. Instead of direct laser exposure, more often than not, optically-trapped beads are attached to the ends or boundaries of the objects for translation, rotation, and stretching. This is referred to as indirect optical manipulation. In this paper, we utilize the concept of robotic gripping to explain the different experimental setups which are commonly used for indirect manipulation of cells, nucleic acids, and motor proteins. We also give an overview of the kind of biological insights provided by this technique. We conclude by highlighting the trends across the experimental studies, and discuss challenges and promising directions in this domain of active current research.

Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors

The use of nucleic acids as components in highly sensitive biosensors has attracted increasing interest during recent years, not least due to the ease by which nucleic acids can be synthesized, manipulated and signal-amplified. To date, several enzymatic reactions, including Polymerase Chain Reaction, Rolling Circle- and Strand Displacement Amplification for signal enhancement of nucleic acid biosensors, have been presented. Of these, the isothermal Rolling Circle Amplification, in which a small single-stranded DNA circle is replicated to generate long tandem repeat products, presents the advantage of allowing single molecule detection and easy quantification as well as of posing little requirement to equipment and personnel training. Rolling Circle Amplification has been used to enhance signals of nucleic acid based sensors specific for a large variety of important biomarkers, including nucleotide sequences, small molecules, proteins and enzyme activities, allowing detection of biomarkers even at the aM concentration level. Moreover, Rolling Circle Products have been adapted to a broad variety of low technological and cost efficient readout formats making the technique appealing for future basic discovery research as well as for at-point-of-care diagnostic or prognostic tests.

Phosphorus and nitrogen interaction: loss of QC identity in response to P or N limitation is antecipated in pdr23 mutant

Changes in root architecture are an important adaptive strategy used by plants in response to limited nutrient availability to increase the odds of acquiring them. The quiescent center (QC) plays an important role by altering the meristem activity causing differentiation and therefore, inducing a determinate growth program. The arabidopsis mutant pdr23 presents primary short root in the presence of nitrate and is inefficient in the use of nucleic acids as a source of phosphorus. In this study the effect of the pdr23 mutation on the QC maintenance under low phosphorus (P) and/or nitrogen is evaluated. QC identity is maintained in wild-type in the absence of nitrate and/or phosphate if nucleic acids can be used as an alternative source of these nutrients, but not in pdr23. The mutant is not able to use nucleic acids efficiently for substitute Pi, determinate growth is observed, similar to wild-type in the total absence of P. In the absence of N pdr23 loses the expression of QC identity marker earlier than wild-type, indicating that not only the response to P is altered, but also to N. The data suggest that the mutation affects a gene involved either in the crosstalk between these nutrients or in a pathway shared by both nutrients limitation response. Moreover loss of QC identity is also observed in wild-type in the absence of N at longer limitation. Less drastic symptoms are observed in lateral roots of both genotypes.

Safe and efficient in vitro and in vivogene delivery: tripodal cationic lipids with programmed biodegradability

The therapeutic use of nucleic acids has long been heralded as a panacea of medicinal opportunity, a vision enhanced by the introduction of RNA interference technology. The Achilles heel of such an approach is the in vivo delivery of the desired nucleic acid into cells, a practice that lacks selectivity, safety and/or efficiency. Herein we report the safe and efficacious in vitro and in vivo delivery of nucleic acids using tripodal biodegradable cationic lipids. Toxicity reduction and transfection potency of these novel amphiphiles were addressed by designing the compounds to undergo complete intracellular degradation thereby enhancing cargo release while minimising toxicity and potential tissue accumulation. Compounds demonstrated high-efficiency in transfecting DNA into cells both in vitro and in vivo with no signs of toxicity, thus potentially offering a safer alternative to viral transfection for gene therapy application.

ChemInform Abstract: Responsive Polyelectrolyte Complexes for Triggered Release of Nucleic Acid Therapeutics

AbstractReview: 85 refs.

Selective gene silencing by viral delivery of short hairpin RNA

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells.

Responsive polyelectrolyte complexes for triggered release of nucleic acid therapeutics

The use of nucleic acids as therapeutics offers many potential benefits for treating disease. However, for these delicate yet potent biomolecules to be practical in the clinic, carrier vehicles are needed not only to protect the nucleic acids during transport in the body, but also release the biopolymers at the disease site. Polycations can meet the complex needs of nucleic acid delivery as they can condense the polyanionic nucleic acids to form stable polyelectrolyte complexes and, through appropriate design, can release the biotherapeutic at a target site. In this feature article, we review recent advances in the field, and indicate the ways in which future materials of this type might lead to enhanced therapies and treatments for currently untreatable diseases.

Structured Nucleic Acid Probes for Electrochemical Devices

AbstractThe use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design.

Relating nucleic acid and protein indices to growth in Mysis relicta: ration, cycling temperature, and metabolism

We investigated growth rate, nucleic acid (DNA, RNA) and protein indices and respira- tion in juvenile (8.5 to 12 mm total body length, 7 to 20 mg wet wt) and young adult (12 to 14 mm, 20 to 30 mg wet wt) Mysis relicta, as a function of temperature, body mass and molt stage in order to develop methods to assess condition or growth in the field. Mysids were exposed to either a preferred temperature (6.5°C) and 3 ration levels, or a range of constant and dielly-cycling (DC) temperatures with ad libitum feeding. Mysid growth parameters (specific rates of growth (SGR), respiration (M O2), and RNA content cell -1 ) integrated the DC temperature experienced as averaged responses weighted by the time spent at each temperature. M O2 peaked at 12.7°C on acute temperature exposure from 4.2°C. MO2 compensation with prolonged temperature exposure occurred at mean diel temperatures ≤8.5°C. Mysids could not survive at 16°C even for 5 h d -1 . These results confirm behavioral observa- tions of temperature preferences. RNA concentration in M. relicta increased with ration and decreas- ing temperatures. Protein:DNA ratio, %protein and SGR increased with ration and then plateaued. Protein:DNA ratio, %protein and DNA:weight ratio did not change with temperature with unlimited feeding. Forward, stepwise, multiple regression models for each experiment and the combined data accounted for 31 to 72% of variability in SGR. Our experimental data provide guidance, a prelimi- nary temperature-correction factor for RNA, and benchmarks for use of nucleic acid and protein indices in assessing growth or condition of M. relicta in the field.

Addressable Molecular Node Assembly - Functional DNA Nanostructures

The use of nucleic acids as a nanomaterial is becoming increasingly widespread due to the suitability of the hydrogen-bonding patterns and sequence specificity inherent to the double-helix. As minimisation of size becomes ever more important it is imperative to employ nucleic acids in the most efficient and functional manner possible. To this end we have constructed DNA nanostructures on what may be the smallest possible scale (basic components of just 10 bp) that not only reliably self-assemble but also where each unit of a 2-dimensional DNA network can be uniquely identified and selectively functionalized.(1,2.3) On this length scale and using full addressability of the network to engrave specific pathways on the scaffold, energy and electron transfer become efficient for potential information storage applications.(4).

Nucleic acid and nucleotide-mediated synthesis of inorganic nanoparticles

Since the advent of practical methods for achieving DNA metallization, the use of nucleic acids as templates for the synthesis of inorganic nanoparticles (NPs) has become an active area of study. It is now widely recognized that nucleic acids have the ability to control the growth and morphology of inorganic NPs. These biopolymers are particularly appealing as templating agents as their ease of synthesis in conjunction with the possibility of screening nucleotide composition, sequence and length, provides the means to modulate the physico-chemical properties of the resulting NPs. Several synthetic procedures leading to NPs with interesting photophysical properties as well as studies aimed at rationalizing the mechanism of nucleic acid-templated NP synthesis are now being reported. This progress article will outline the current understanding of the nucleic acid-templated process and provides an up to date reference in this nascent field.

Optimization of Ribonucleic Acid Detection From Archival Guinea Pig Temporal Bone Specimens

The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

A Bifunctional Δ12,Δ15-Desaturase from Acanthamoeba castellanii Directs the Synthesis of Highly Unusual n-1 Series Unsaturated Fatty Acids

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.

DNA Unwinding by Escherichia coli DNA Helicase I (TraI) Provides Evidence for a Processive Monomeric Molecular Motor

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

Nucleic Acids and Their Analogs as Nanomaterials for Biosensor Development

Nucleic acids are natural biopolymers that store the genetic information of organisms. This makes the detection and characterization of DNA and RNA a relevant task in biotechnology, with applications ranging from medicine to environmental control. During the last decades, a large effort has been focused on the development of biosensors, among them those devoted to the detection of nucleic acids in natural samples and those that include nucleic acids as nanosized capture probes for different biomolecules. DNA microarray technology has been successfully used in biotechnological applications including genotyping and gene expression studies. Nevertheless, the performance of DNA microarrays has a limitation imposed by the need of a previous fluorescent labeling of the target molecule to be analyzed. This encouraged the use of alternative detection methods, such as optical and electrochemical ones, and recently others based on surface characterization techniques. New trends in nanotechnology point towards new tools for manipulating molecules and macromolecules that could be developed as high performance biosensors. This interdisciplinary approach towards the integration of novel biosensors can benefit from the capability of certain polymers to form self-assembled monolayers (SAMs) on different surfaces. Thiol-modified DNA can form SAMs on gold surfaces with reduced efficiency, and the biological activity of the probe is decreased upon adsorption. Therefore, thiolated DNA has a very limited use in biosensor development. These constraints have been successfully by-passed using uncharged, artificial analogs of natural nucleic acids, such as peptide nucleic acids (PNAs), as molecular probes. This contribution reviews the state of the art in the use of nucleic acids and their analogs as biosensor nanomaterials, and summarizes the novel approach towards the development of biosensors based on SAMs of PNAs. Finally, we present the current trends in this promising aspect of nanobiotechnology.