Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue.

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M-1cm-1 in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives.

A peptide nucleic acid (PNA) heteroduplex probe containing an inosine-cytosine base pair discriminates a single-nucleotide difference in RNA.

Selective discrimination of a single-nucleotide difference in single-stranded DNA or RNA remains a challenge with conventional DNA or RNA probes. A peptide nucleic acid (PNA)-derived probe, in which PNA forms a pseudocomplementary heteroduplex with inosine-containing DNA or RNA, effectively discriminates a single-nucleotide difference in a closely related group of sequences of single-stranded DNA and/or RNA. The pseudocomplementary PNA heteroduplex is easily converted to a fluorescent probe that distinctively detects a member of highly homologous let-7 microRNAs.

Measurement of telomere length using PNA probe by cytometry.

Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo-PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide.

Locked nucleic acid based beacons for surface interaction studies and biosensor development.

DNA sensors and microarrays permit fast, simple, and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective, and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon's poor stability because of the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics, and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity, and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization.

Synthesis of a fluorescent 2'3'-dideoxycytosine analog, tCdd.

The pathway leading to the preparation of a novel tricyclic 2'3'-dideoxycytosine analog, tCdd (1) is reported. A protected 2'3'-dideoxyribose prepared from l-glutamic acid was coupled to a silylated fluorescent base to yield a mixture of the alpha- and beta-anomers of the 2'3'-dideoxyribonucleoside of 1,3-diaza-2-oxophenothiazine, tCdd (1). The fluorescent base analog retains a high fluorescence emission over a large pH range and should be useful in a variety of probe applications.

Synthesis and properties of a novel molecular beacon containing a benzene-phosphate backbone at a stem moiety.

This paper describes the synthesis and properties of a novel molecular beacon (MB) containing a benzene-phosphate backbone at the stem moieties. Fluorescent intensity of MBs was found to be stabilized by introducing a benzene-phosphate backbone at stem moieties.

Synthesis and oligonucleotide incorporation of fluorescent cytosine analogue tC: a promising nucleic acid probe.

The tricyclic cytosine, tC, is a fluorescent base analogue with excellent properties for investigating intrinsic characteristics of nucleic acid as well as interactions between nucleic acids and other molecules. Its unique fluorescence properties and insignificant influence on overall structure and dynamics of nucleic acid after incorporation makes tC particularly interesting in fluorescence resonance energy transfer and anisotropy measurements. We here describe a straightforward synthesis of the standard monomer form of tC for DNA solid-phase synthesis, the tC phosphoramidite, and its subsequent incorporation into oligonucleotides. The total synthesis of the tC phosphoramidite takes approximately 8 days and its incorporation and the subsequent oligonucleotide purification an additional day.

Synthesis, characterization and hybridization studies of new nucleo-gamma-peptides based on diaminobutyric acid.

In the present work, we report the synthesis and the characterization of a new chiral nucleoaminoacid, in which a diaminobutyric moiety is connected to the DNA nucleobase by an amidic bond, and its oligomerization to give the corresponding nucleo-gamma-peptide. The ability of this synthetic polymer to bind complementary DNA was studied in order to explore its possible use in antigene/antisense or diagnostic applications. Our interest in the presented DNA analogue was also supported by the importance of gamma-aminoacid-containing compounds in natural products of biological activity and by the known stability of gamma-peptides to enzymatic degradation. Furthermore, our work could contribute to the study of the role of nucleopeptides as prebiotic material in a PNA world that could successively lead to the actual DNA/RNA/protein world, as recently assumed.

High-throughput real-time quantitative reverse transcription PCR.

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Construction of a novel peptide nucleic acid piezoelectric gene sensor microarray detection system.

A novel 2 x 5 clamped style piezoelectric gene sensor microarray has been successfully constructed. Every crystal unit of the fabricated gene sensor can oscillate independently without interfering with each other. The bis-peptide nucleic acid (bis-PNA) probe, which can combine with target DNA or RNA sequences more effectively and specifically than a DNA probe, was designed and immobilized on the surface of the gene sensor microarray to substitute the conventional DNA probe for direct detection of the hepatitis B virus (HBV) genomic DNA. Detection conditions were then explored and optimized. Results showed that PBS buffer of pH 6.8, an ion concentration of 20 mmol/liter, and a probe concentration of 1.5 micromol/liter were optimal for the detection system. Under such optimized experimental conditions, the specificity of bis-PNA was proved much higher than that of DNA probe. The relationship between quantity of target and decrease of frequency showed a typical saturation curve when concentrations of target HBV DNA varied from 10 pg/liter to 100 microg/liter, and 10 microg/liter was the watershed, with a statistic linear regression equation of I gC = -2.7455 + 0.0691 deltaF and the correlating coefficient of 0.9923. Fortunately, this is exactly the most ordinary variant range of the HBV virus concentration in clinical hepatitis samples. So, a good technical platform is successfully constructed and it will be applied to detect HBV quantitatively in clinical samples.

High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides.

DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.

PNA microarrays for hybridisation of unlabelled DNA samples.

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces.

Platinum(II)-based coordination compounds as nucleic acid labeling reagents: synthesis, reactivity, and applications in hybridization assays.

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5'-monophosphate (5'-GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic approximately aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2 x 10(-2) M(-1) s(-1)) and the slowest reacting complex (k=0.93 x 10(-2) M(-1) s(-1)) under these conditions. Platination of an oligodeoxynucleotide (30-mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.

A dumbbell-like complex formation for DNA target assay.

A simple and highly sensitive test for the detection of nucleic acid targets is described. It is based upon complex formation between a small-diameter magnetic particle and a larger and nonmagnetic particle through a hybridization reaction, what we have called a dumbbell-like complex. During the different steps, nonreacting nonmagnetic conjugates were eliminated by magnetic separation. At the end of the process, dumbbell complex number was estimated by counting under a microscope. Compared to the already described two-particle tests, our model was able to reach higher sensitivities, with a threshold typically in the amol/mL range (10(6) copies of HIV DNA/mL) without the need for complex instrumentation or genomic amplification reactions.

Synthesis and binding properties of oligo-2'-deoxyribonucleotides covalently linked to a thiazole orange derivative.

Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2'-deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the alpha and the beta series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2'-deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-alpha-thymidylate or oligo-beta-thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.

Real-time monitoring of intracellular mRNA hybridization inside single living cells.

A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells.

Using the polymerase chain reaction to maintain DNA probe inventories in clinical and diagnostic laboratories.

Clinical and diagnostic DNA laboratories must maintain a large inventory of DNA probes for use in hybridization studies. The preparation of plasmid DNA and isolation of DNA fragments for use as probes in both expensive and time consuming. We present here a rapid and relatively inexpensive method of producing large amounts of DNA fragments from stocks, using the polymerase chain reaction (PCR). Our experience over the past year using this technique has been very positive and we believe many laboratories could benefit by employing such a labor-saving approach to maintaining DNA probes. The technique uses the bacteriophage M13 DNA sequencing primers to amplify cloned inserts contained in commonly used plasmid vectors. As examples, we illustrate the use of DNA produced in this manner as probes for linkage analysis of the fragile X syndrome and for detection of deletions in the Duchenne muscular dystrophy gene. We have also found that at least two probes can be amplified in the same PCR reaction, allowing the detection of two different restriction fragment length polymorphisms (RFLP) simultaneously. It should be possible for laboratories to devise strategies particular to their individual needs using more than one DNA probe produced in the same PCR reaction to detect RFLP's. Such strategies would need only to consider that the predicted alleles of the multiple polymorphisms do not migrate to the same position during electrophoresis. Stocks of single or multiple probes produced by the PCR could then be maintained for more rapid Southern analyses.