Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

Complexes of nucleolipid liposomes with single-stranded and double-stranded nucleic acids

We report on the association of anionic liposomes from POP-Ade:POPC (1-palmitoyl-2-oleoyl-phosphatidyladenosine and 1-palmitoyl-2-oleoyl-phosphatidylcholine, respectively) with single- and double-strand nucleic acids, mediated by Ca(2+) bridging. The structural and dynamical features of such complexes are compared with those displayed when the nucleolipid is replaced by POPG (1-palmitoyl-2-oleoyl-sn-phosphatidyl-glycerol), characterized by the same apolar skeleton and negative charge as POP-Ade, but lacking the nucleic polar head. For single-stranded nucleic acids, we demonstrate that specific interactions drive the formation of complexes with nucleolipid liposomes, while no association is present for POPG-based samples. For double-stranded nucleic acids, Ca(2+) bridging promotes association with both liposomal formulations, but the corresponding complexes have different structural features, in terms of size, overall charge and internal liquid-crystalline structure.     

Liquid crystalline dispersions of complexes formed by chitosan with double-stranded nucleic acids

Double-stranded molecules of nucleic acids (NAs) were shown to interact with chitosans to form under certain conditions (chitosan molecular mass, content of amino groups, distance between amino groups, pH of solution, etc.) multiple types of liquid crystalline dispersions. The dispersions formed are different in their spatial structures, and hence in the sense and magnitude of the abnormal optical activity. The physicochemical properties of these dispersions were investigated. Time- and temperature-stabilization of dispersions that possess abnormal optical activity were achieved by chemical crosslinking of chitosan molecules in the liquid crystalline dispersions formed from NA–chitosan complexes. The accessibility of these ‘NA–liquid crystalline elastomers' with respect to enzyme and drug action was tested. The multiplicity of liquid crystalline forms of DNA–chitosan complexes was possibly explained by the influence of the character of the dipole distribution over the surface DNA molecules on the sense of the spatial twist of the cholesteric liquid crystalline dispersions resulting from these complexes.     

Liquid crystalline dispersions of complexes formed by chitosan with double-stranded nucleic acids

Double-stranded molecules of nucleic acids (NAs) were shown to interact with chitosans to form under certain conditions (chitosan molecular mass, content of amino groups, distance between amino groups, pH of solution, etc.) multiple types of liquid crystalline dispersions. The dispersions formed are different in their spatial structures, and hence in the sense and magnitude of the abnormal optical activity. The physicochemical properties of these dispersions were investigated. Time- and temperature-stabilization of dispersions that possess abnormal optical activity were achieved by chemical crosslinking of chitosan molecules in the liquid crystalline dispersions formed from NA-chitosan complexes. The accessibility of these 'NA-liquid crystalline elastomers' with respect to enzyme and drug action was tested. The multiplicity of liquid crystalline forms of DNA-chitosan complexes was possibly explained by the influence of the character of the dipole distribution over the surface DNA molecules on the sense of the spatial twist of the cholesteric liquid crystalline dispersions resulting from these complexes.     

A novel type of microscopic size chip based on double-stranded nucleic acids

The double-stranded molecules of nucleic acids (NA) of B- and A-families fixed in the structure of cholesteric liquid-crystalline dispersions, formed as a result of phase exclusion of these molecules from polymer-containing solution, have been used as 'building blocks' for the molecular design. Using the formation of polymeric chelate bridges between NA molecules, three-dimensional structures consisting of alternating NA, anthracycline and copper ions, were created. The formation of the polymeric chelate bridges allows one to stabilize the initial spatial mode of ordering of neighboring NA molecules in a form of so-called 'molecular constructions', immobilize these constructions onto supporting film and evaluate their sizes and shape. The creation of NA molecular constructions is accompanied by an 'extra-increase' in the amplitude of the bands in the CD spectra, despite the initial sense of cholesteric twisting characteristic of liquid-crystalline dispersions. Destroying of polymeric chelate bridges between NA molecules by action of biologically relevant compounds results in disintegration of NA liquid-crystalline molecular constructions. Three-dimensional NA molecular construction can be used as a microscopic size multifunctional chemical unit (chip) for biological or chemical needs.     

Electrochemical sensor for detection of unmodified nucleic acids

We have developed a nucleic acid (NA) sensor based on mediated electrochemical oxidation of guanine residues. In this method, oligonucleotide probes are bound to a tin-doped indium oxide (ITO) electrode through a self-assembled phosphonate monolayer. The end carboxyl moiety of the monolayer is activated with carbodiimide and reacted with the amine group of a C6 alkyl linker which has been added to the 5'-end of the oligonucleotide probe. Upon hybridization of the complementary target NA, the hybrid is detected using a redox-active mediator, tris(2,2'-bipyridyl) ruthenium(II). We speculate that the monolayer does not impede electron-transfer since it contains many defect sites when assembled on a polycrystalline ITO surface. These defect sites are accessible to the mediator, but not to NA or proteins. The electrocatalytic current was a linear function of the amount of guanine bound at the electrode surface, with a detection limit of 120 amoles of guanine cm(-2) at 0.28 cm(2) ITO electrodes.     

2'-Ribose-ferrocene oligonucleotides for electronic detection of nucleic acids

We have synthesized two novel phosphoramidites with a ferrocenyl moiety at the 2'-ribose position linked through a butoxy linker. Using automated DNA/RNA synthesis techniques, oligonucleotides containing ferrocene at various positions were prepared and characterized by HPLC, MALDI-TOF mass spectrometry, and electrochemistry. Thermal stability studies of the ferrocene-modified DNA duplexes revealed that introduction of one or two ferrocenyl complexes does not result in an observed change of the T(m) values of the corresponding DNA duplexes when compared to the nonmodified hybrids. These data indicate that the introduction of a ferrocenyl group at the 2'-position of the ribose ring containing either a purine or pyrimidine base has no effect on the stability of the modified DNA. The electrochemical behavior of the ferrocene-containing DNA was examined by cyclic voltammetry. The modified 2'-ferrocene-oligonucleotides are electrochemically active and can be used as signaling probes for the electronic detection of nucleic acids on bioelectronic sensors.     

High-throughput ribozyme-based assays for detection of viral nucleic acids

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.     

Enzyme-free and multiplexed microRNA detection using microRNA-initiated DNA molecular motor

In this work, we have developed a sensitive, simple, and enzyme-free assay for detection of microRNAs (miRNAs) by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains. In the presence of miRNA target, it can hybridize with one of the stem-loop DNA to open the stem and to produce a miRNA/DNA hybrid and a single strand (ss) DNA, the ssDNA will in turn hybridize with another stem-loop DNA and finally form a double strand (ds) DNA to release the miRNA. One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence. The formation of dsDNA can produced specific fluorescence signal for miRNA detection. The released miRNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor, which results in great fluorescence amplification. With the efficient signal amplification, as low as 1 pmol/L miRNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained. Moreover, by designing different stem-loop DNAs specific to different miRNA targets and labeling them with different fluorophores, multiplexed miRNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum (SFS) technique.     

Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.     

Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence

In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of “split” G-quadruplex probes on hybridization with an arbitrary “target” DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex–quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any “target” DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed “turn-off” whereas the biosensors containing NMM are defined as “turn-on”. Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H₂O₂, hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.     

Homogenous rapid detection of nucleic acids using two-color quantum dots

We report a homogenous method for rapid and sensitive detection of nucleic acids using two-color quantum dots (QDs) based on single-molecule coincidence detection. The streptavidin-coated quantum dots functioned as both a nano-scaffold and as a fluorescence pair for coincidence detection. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary target DNA through a sandwich hybridization reaction. The DNA hybrids were first caught and assembled on the surface of 605 nm-emitting QDs (605QDs) through specific streptavidin-biotin binding. The 525 nm-emitting QDs (525QDs) were then added to bind the other end of DNA hybrids. The coincidence signals were observed only when the presence of target DNA led to the formation of 605QD/DNA hybrid/525QD complexes. In comparison with a conventional QD-based assay, this assay provided high detection efficiency and short analysis time due to its high hybridization efficiency resulting from the high diffusion coefficient and no limitation of temperature treatment. This QD-based single-molecule coincidence detection offers a simple, rapid and ultra sensitive method for genomic DNA analysis in a homogenous format.     

Binary malachite green aptamer for fluorescent detection of nucleic acids

A new probe that can fluorescently report the presence of specific nucleic acids in solution with extremely high selectivity was developed. The probe consists of malachite green-a triphenylmethane dye-and two short RNA strands, each of which comprises a fragment complementary to an analyte molecule and a fragment of a malachite green aptamer (MGA). The two RNA strands form MGA upon hybridization to the adjacent positions of the nucleic acid analyte. MGA is able to bind malachite green and enhance the fluorescence of the dye, thus monitoring the presence of the nucleic acid in solution. The probe reliably discriminates against 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at room temperature in physiological buffer. Consisting of unmodified RNA strands, which can be expressed in living cells, binary MGA probe represents a promising instrument for real-time nucleic acid monitoring in vivo.     

Direct molecular detection of nucleic acids by fluorescence signal amplification

An integrated PCR-free DNA sensor, which combines a sequence-specific receptor, an optical polymeric transducer, and an intrinsic fluorescence amplification mechanism, is reported. This sensor is based on the different conformations adopted by a cationic polythiophene when electrostatically bound to ss-DNA or ds-DNA, and on the efficient and fast energy transfer between the resulting fluorescent polythiophene/ds-DNA complex and neighboring fluorophores attached to ss-DNA probes. This molecular system allows the detection of only five molecules in 3 mL of an aqueous solution, or 3 zM, in 5 min. Moreover, this work demonstrates, for the first time, the direct detection of single nucleotide polymorphisms (SNPs) from clinical samples in only a few minutes, without the need for nucleic acid amplification.     

Multiplexed detection of nucleic acids in a combinatorial screening chip

Multiplexed diagnostic testing has the potential to dramatically improve the quality of healthcare. Simultaneous measurement of health indicators and/or disease markers reduces turnaround time and analysis cost and speeds up the decision making process for diagnosis and treatment. At present, however, most diagnostic tests only provide information on a single indicator or marker. Development of efficient diagnostic tests capable of parallel screening of infectious disease markers could significantly advance clinical and diagnostic testing in both developed and developing parts of the world. Here, we report the multiplexed detection of nucleic acids as disease markers within discrete wells of a microfluidic chip using molecular beacons and total internal reflection fluorescence microscopy (TIRFM). Using a 4 × 4 array of 200 pL wells, we screened for the presence of four target single stranded oligonucleotides encoding for conserved regions of the genomes of four common viruses: human immunodeficiency virus-1 (HIV-1), human papillomavirus (HPV), Hepatitis A (Hep A) and Hepatitis B (Hep B). Target oligonucleotides are accurately detected and discriminated against alternative oligonucleotides with different sequences. This combinatorial chip represents a versatile platform for the development of clinical diagnostic tests for simultaneous screening, detection and monitoring of a wide range of biological markers of disease and health using minimal sample size.     

First improvements in the detection and quantification of label-free nucleic acids by laser-induced breakdown spectroscopy: Application to the deoxyribonucleic acid micro-array technology

The accurate quantification of nucleic acids is essential in many fields of modern biology and industry, and in some cases requires the use of fluorescence labeling. Yet, in addition to standardization problems and quantification reproducibility, labeling can modify the physicochemical properties of molecules or affect their stability. To address these limitations, we have developed a novel method to detect and quantify label-free nucleic acids. This method is based on stoichiometric proportioning of phosphorus in the nucleic acid skeleton, using laser-induced breakdown spectroscopy, and a specific statistical analysis, which indicates the error probability for each measurement. The results obtained appear to be quantitative, with a limit of detection of 10⁵ nucleotides/µm² (i.e. 2 × 10¹³ phosphorus atoms/cm²). Initial micro-array analysis has given very encouraging results, which point to new ways of quantifying hybridized nucleic acids. This is essential when comparing molecules of different sequences, which is presently very difficult with fluorescence labeling.     

Chemical ligation as a method for the assembly of double-stranded nucleic acids: Modifications and local structure studies

A new procedure was developed as an alternative to the enzymatic assembly of natural and modified double-stranded DNAs using chemical reagent (chemical ligation). BrCN was suggested as an efficient coupling reagent, which induces superfast reactions in DNA duplexes. The physicochemical properties and the structure of new types of DNA duplexes, which are the substrates for chemical ligation, with breaks in phosphodiester chains, including concatemers, were studied. Chemical ligation was applied to prepare biologically active 17–200 base-pair double-stranded DNAs and DNA-RNA block-copolymers, to incorporate various modifications into DNA duplexes including pyrophosphate and phosphoramidate unnatural internucleotide bonds. The unique possibilities of this approach were demonstrated in the development of methods for circularization of oligodeoxy ribonucleotides and assembly of branched DNAs. The structural-kinetic concept of chemicalligation was created and the relationship between the reactivity of interacting groups and sequence-dependent local conformation of the ligation site in B-DNA was established. The lesser efficiency of chemical ligation of RNA fragments in comparison to that of DNA analogs was demonstrated and rationalized. This approach was used as a sensitive monitor of a stable double helix formation and third-strand binding to a DNA duplex.Translated from Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1889–1911, August, 1996.     

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5′-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G_n (n ≥ 2) repeat was extended from its 3′-end. This G_n repeat, together with the three GGG repeats at the 5′-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.     

Dissecting the strand folding orientation and formation of G-quadruplexes in single- and double-stranded nucleic acids by ligand-induced photocleavage footprinting

The widespread of G-quadruplex-forming sequences in genomic DNA and their role in regulating gene expression has made G-quadruplex structures attractive therapeutic targets against a variety of diseases, such as cancer. Information on the structure of G-quadruplexes is crucial for understanding their physiological roles and designing effective drugs against them. Resolving the structures of G-quadruplexes, however, remains a challenge especially for those in double-stranded DNA. In this work, we developed a photocleavage footprinting technique to determine the folding orientation of each individual G-tract in intramolecular G-quadruplex formed in both single- and double-stranded nucleic acids. Based on the differential photocleavage induced by a ligand tetrakis(2-trimethylaminoethylethanol) phthalocyaninato zinc tetraiodine (Zn-TTAPc) to the guanines between the two terminal G-quartets in a G-quadruplex, this method identifies the guanines hosted in each terminal G-quartets to reveal G-tract orientation. The method is extremely intuitive, straightforward, and requires little expertise. Besides, it also detects G-quadruplex formation in long single- and double-stranded nucleic acids.     

Label-free visual detection of nucleic acids in biological samples with single-base mismatch detection capability

We have combined an allosteric molecular beacon for target recognition and guanine-rich DNAzyme for signal amplification to develop a new platform for visual detection of nucleic acids with single-base mismatch detection capability. The fully DNA-structured platform can undergo color change in response to target DNA/RNA, which enables sensitive and selective visual detection in biological samples.