Noncovalent Spin‐Labeling of DNA and RNA Triplexes

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine‐derived label and triplex motifs containing a single‐nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.     

Spin labeled nucleic acids.

Homopolyribonucleotides and E. coli DNA wer spin labeled with an iodoacetamide-nitroxide compound. The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid. This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers. In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations. An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal. The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs). These critical temperatures are lower than the optical Tm. Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.     

Peptide nucleic acid-assisted topological labeling of duplex dna

Peptide nucleic acids (PNAs) are a family of synthetic polyamide mimics of nucleic acids that offer a variety of applications. Pyrimidine bis-PNAs can be used for rational design of novel interlocked DNA nanostructures, earring labels, representing locked pseudorotaxanes or locked catenanes. These structures are created through DNA ligase-mediated catenation of duplex DNA with a circularized oligonucleotide tag at a designated DNA site. The assembly is performed via formation of the PD-loop consisting of a pair of bis-PNA openers and the probe oligonucleotide. The openers locally expose one of the two strands of duplex DNA for hybridizing the probe, whose termini are complementary to the displaced DNA strand. After hybridization, they are in juxtaposition and can subsequently be linked by DNA ligase. As a result, a true topological link forms at a precise position on the DNA double helix yielding locked, earring-like label. DNA topological labeling can be done both in solution and, for longer templates, within the agarose gel plug. Accordingly, highly localized DNA detection with rolling circle amplification of hybridization signal and effective micromanipulations with DNA duplexes become possible through precise spatial positioning of various ligands on the DNA scaffold.     

Kinase-catalyzed biotinylation of DNA

Prior work documented use of γ-phosphate modified ATP analogs to label DNA using T4 polynucleotide kinases (T4PNK), although applications have been limited. To fully characterize kinase-catalyzed labeling of nucleic acids, we explored use of ATP-biotin as a cosubstrate with T4PNK. T4PNK accepted ATP-biotin to 5′-label single stranded DNA. However, T4PNK-mediated labeling of double stranded substrates was low yielding. In addition, the phosphoramidate bond connecting the biotin group to the DNA was unstable. These results suggest that kinase-catalyzed biotinylation will be useful with single stranded DNA substrates and mild reaction conditions. By revealing the scope and limitations of kinase-catalyzed biotinylation, these studies provide a foundation for future development and application of kinase-catalyzed labeling to DNA-based biological studies.     

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:a-2',3':c]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.     

Increasing the luminescence of lanthanide complexes.

This review compares the chemical and physical properties of lanthanide ion complexes and of other narrow-emitting species that can be used as labels for cytometry. A series of luminescent lanthanide ion macrocyclic complexes, Quantum Dyes, which do not release or exchange their central lanthanide ion, do accept energy transfer from ligands, and are capable of covalent binding to macromolecules, including proteins and nucleic acids, is described and their properties are discussed.Two methods are described for increasing the luminescence intensity of lanthanide ion complexes, which intrinsically is not as high as that of standard fluorophores or quantum dots. One method consists of adding a complex of a second lanthanide ion in a micellar solution (columinescence); the other method produces dry preparations by evaporation of a homogeneous solution containing an added complex of a second lanthanide ion or an excess of an unbound antenna ligand. Both methods involve the Resonance Energy Transfer Enhanced Luminescence, RETEL, effect as the mechanism for the luminescence enhancement.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.     

DNA Probes: Applications of the Principles of Nucleic Acid Hybridization

Abstract

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.     

DNA probes: applications of the principles of nucleic acid hybridization.

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02–1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5′-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.     

The influence of polyamine-nucleic acid complexes on Fe2+ autoxidation

Summary Polyamines are able to affect Fe²⁺ autoxidation in the presence of suitable low molecular weight phosphorus-containing compounds; the inhibitory effect exerted by polyamines is directly related to their ability to bind phosphorus-containing compounds [1].It is well known that polyamines, as polycations at physiological pH, bind strongly to nucleic acids. In this paper it is shown that polyamines, also in the presence of nucleic acids, inhibit Fe²⁺ autoxidation and thus depress the generation of free oxygen radicals. Most of the nucleic acids tested inhibited Fe²⁺ autoxidation although the concentration which causes half maximal effect differs. Polyamine effect on Fe²⁺ autoxidation varies greatly depending on the single or double stranded nature of the nucleic acid. In the present of single stranded nucleic acids, spermine and spermidine potentiate the inhibition of Fez+ autoxidation by these nucleic acids. A relationship exists between the ability of spermine to interact with single stranded nucleic acids and to inhibit Fe²⁺ autoxidation in their presence. When double stranded nucleic acids are present, polyamines reverse the inhibition of Fee+ autoxidation exerted by these nucleic acids. Molecular mechanisms are proposed to explain these experimental results. The hypothesis that polyamines may inhibit oxidative damage caused to nucleic acids by Fe²⁺ autoxidation, is also discussed.Abbreviations poly [A] polyadenylic acid (5) - poly [C] polycytidylic acid (5) - poly [1] polyinosinic acid (5) - poly [G] polyguanylic acid (5) - poly [A. U] polyadenylic-uridylic acid - poly [A] poly [U] polyadenylic-polyuridylic acid     

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms.We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.     

Conjugated polyelectrolytes for label-free DNA microarrays

Classical strategies for gene microarrays require labeling of probes or target nucleic acids with signaling molecules, a process that is expensive, time consuming and not always reliable. Bazan and colleagues showed that a nucleic acid-binding cationic conjugated polyelectrolyte can be used in label-free DNA microarrays based on surfaces modified with neutral peptide nucleic acid (PNA) probes. This technique provides a simple and sensitive method for DNA detection without the need for covalent labeling of target DNA.     

Length does matter for cGAS

Recognition of foreign nucleic acids by the immune system is essential to host protection against many viral and bacterial infections. It relies on the capacity of innate immune sensors to selectively distinguish self‐ and non‐self‐nucleic acids, on the basis of a variety of parameters including base modifications, sequence composition, length or subcellular localisation. In this issue of EMBO Reports, Luecke et al 1 describe that the sensing of cytoplasmic double‐stranded DNA by the cyclic GMP–AMP (cGAMP) synthase (cGAS) is much more sensitive for longer fragments, when low doses of cytoplasmic DNA are used. This finding repositions length as the predominant factor governing the discrimination between self‐ and non‐self‐cytoplasmic DNA.     

Recent advances in fluorescence sensors based on DNA–MOF hybrids

In this review, the recent advances in the development of fluorescence sensors based on DNA and metal–organic framework hybrids have been reported for nucleic acid, metal ion and amino acid detection. The main detection mechanism depends on different adsorption capacities of MOFs towards different DNA structures (single‐stranded DNA, double‐stranded DNA), and consequently the fluorescence intensity of probe DNA is changed. These results might open up a way to study their potential application in material science and clinical diagnosis of some related diseases.     

Cyanine dyes for the detection of double stranded DNA

Twenty three novel cyanine dyes have been applied as fluorescent stains for the detection of nucleic acids in agarose gel electrophoresis. Significant fluorescence enhancement of these dyes in the presence of double stranded DNA was observed. Five dyes offered superior sensitivity in the detection and quantification of DNA, over Ethidium Bromide, the most commonly used nucleic acid stain.     

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.     

Universal labeling chemistry for nucleic acid detection on DNA-arrays

We show here a new and efficient aqueous chemistry for labeling of any class of nucleic acids for their detection on DNA chip. The labels contain a diazo function as reactive moiety and biotin as detectable unit. The highly selective reaction of diazo group on the phosphate does not disrupt base pairing recognition and hybridization specificity.