Circulating nucleic acids (CNAs) and cancer—A survey

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.     

Development of a sensitive and rapid nucleic acid assay with tetraphenyl porphyrinatoiron chloride by a resonance light scattering technique.

Based on the interaction between nucleic acids and tetraphenyl porphyrinatoiron chloride (FeTPPCl), a novel method for the determination of nucleic acids at the nanogram level has been developed. Under the optimum conditions, the weak resonance light scattering (RLS) intensity of FeTPPCl was greatly enhanced by nucleic acids and the enhanced RLS intensity was proportional to the concentration of nucleic acids in the range 0.02-2.8 microg/mL for calf thymus DNA, 0.05-3.3 microg/mL for fish sperm DNA and 0.07-4.5 microg/mL for yeast RNA. The detection limits (3sigma) were 2.9 ng/mL for calf thymus DNA, 3.9 ng/mL for fish sperm DNA and 5.2 ng/mL for yeast RNA. Almost no interference could be observed from proteins, nucleosides and most of the metal ions. The proposed method showed good reliability, sensitivity, rapidity and reproducibility when applied to the determination of nucleic acids in synthetic and biochemical samples. The time savings make this method suitable for large routine analyses.     

Factors contributing to the outcome of oxidative damage to nucleic acids

Oxidative damage to DNA appears to be a factor in cancer, yet explanations for why highly elevated levels of such lesions do not always result in cancer remain elusive. Much of the genome is non-coding and lesions in these regions might be expected to have little biological effect, an inference supported by observations that there is preferential repair of coding sequences. RNA has an important coding function in protein synthesis, and yet the consequences of RNA oxidation are largely unknown. Some non-coding nucleic acid is functional, e.g. promoters, and damage to these sequences may well have biological consequences. Similarly, oxidative damage to DNA may promote microsatellite instability, inhibit methylation and accelerate telomere shortening. DNA repair appears pivotal to the maintenance of genome integrity, and genetic alterations in repair capacity, due to single nucleotide polymorphisms or mutation, may account for inter-individual differences in cancer susceptibility. This review will survey these aspects of oxidative damage to nucleic acids and their implication for disease.     

Circulating nucleic acids as a tumor marker

Patients suffering from malignant diseases have been shown to have increased amounts of cell free nucleic acids in their circulation. As genetic and epigenetic alterations are increasingly characterized in different types of tumors, such changes can be used to detect tumor-derived nucleic acids in the circulation. To date, nearly all tumor-associated nucleic acids have been detected in the plasma or serum of cancer patients. Moreover, increased levels of circulating viral nucleic acids have also been demonstrated in patients with certain cancers associated with viral infections. The concentration of these tumor-associated nucleic acid species is generally related to the tumor load and the extent of the disease. Serial monitoring of plasma nucleic acids thus provides a good way to follow disease progress and to predict the outcome of such patients. In this review, different approaches of detecting tumor-related nucleic acids in the circulation and their potential as tumor markers in the screening, monitoring and prognostication of malignant diseases are discussed.     

Extracellular nucleic acids

Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.     

Postcolumn nucleic acid intercalation for the fluorescent detection of nucleic acids using ion pair reverse phase high-performance liquid chromatography

Here we report a simple and effective procedure enabling the fluorescent detection of nucleic acids following the rapid, high-resolution separation using ion pair reverse phase chromatography. This approach uses postcolumn nucleic acid intercalation of fluorescent dyes with subsequent fluorescent detection, demonstrating more than a 1000-fold increase in sensitivity in the detection of nucleic acids when compared with traditional UV detection. Moreover, a wide range of intercalating dyes can be incorporated, including those known to disrupt the structure of the nucleic acids, thereby enabling the sensitive detection of DNA and RNA with no adverse effect on resolution of the nucleic acids during ion pair reverse phase chromatography. In addition, such approaches allow one to readily distinguish single-stranded DNA from double-stranded DNA following their separation using ion pair reverse phase high-performance liquid chromatography.     

Effects of the trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds.Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated.Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI).The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology

During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide chemistry. Within the past year, significant progress in in situ hybridisation technology has been achieved, which has resulted, in particular, in reliable and sensitive methods for detection of bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction clamping method have been expanded, and novel ways of exploiting complexes of peptide nucleic acids with double-stranded DNA, such as double duplex invasion complexes and PD loops, have been developed.     

Nucleic acid aptamers and enzymes as sensors

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.     

Novel nucleic acid detection strategies based on CRISPR-Cas systems: From construction to application

Beyond their widespread application as genome-editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR-Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR-Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.     

Fractionation of nucleic acids with special regard to rapidly labeled RNA.

Fractionation of nucleic acids with potassium acetate is useful for studying the synthesis of ribosomal RNA and messenger RNA. By quantitative removal of 4 and 5 s RNA and of DNA, the detection of messenger RNA is facilitated.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.     

Selective staining by 4'', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Hybridization histochemistry or in situ hybridization in the study of neuronal gene expression

1. Currently popular techniques of in situ hybridization histochemistry for the detection of cellular nucleic acids (DNA or RNA) are reviewed. 2. The advantages of single stranded DNA or RNA probes are discussed, together with the advantages of radioactive versus non-radioactive detection of nucleic acid signal. 3. Improving techniques of non-radioactive labelling and the use of image analysis for quantitation of radioactive signals will greatly expand the use of in situ techniques which will become commonplace in the laboratory.     

SNP与基因检测及表达中的RCA技术

滚环扩增(rollingcircleamplification,RCA)技术是一种新的分子生物学检测方法。该方法不仅可以在体外等温条件下对核酸进行高度特异性的检测,而且还可通过线性或指数扩增来进行信号级联放大,其灵敏度能达到1个拷贝的核酸分子,因此,可用于痕量分子的检测。目前,滚环扩增技术广泛应用于全基因组DNA检测、核酸测序、单核苷酸多态性、DNA芯片及蛋白质芯片分析等领域。