Top PDF Functional nucleic acid probes and uses thereof

Functional nucleic acid probes and uses thereof

Functional nucleic acid probes and uses thereof

The present invention provides functional nucleic acid probes, and methods of using functional nucleic acid probes, for binding a target to carry out a desired function. The probes have at least one functional nucleic acid, at least one regulating nucleic acid, and at least one attenuator. The functional nucleic acid is maintained in an inactive state by the attenuator and activated by the regulating nucleic acid only in the presence of a regulating nucleic acid target. In its activated state the functional nucleic acid can bind to its target to carry out a desired function, such as generating a signal, cleaving a nucleic acid, or catalyzing a reaction.
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Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

Next, we examined the potential for the use of DNA-based helper probes in a hybrid DNA/PNA system. PNA synthesis is expensive, and while it may be possible to use unlabeled PNA probes as helpers to increase the accessibility of the target region, this would be cost-prohibitive. Although the low-salt conditions for PNA hybridization are not optimal for DNA-FISH, they are similar to those used for PCR, a DNA-based, hybridization-de- pendent process that typically uses 50 mM KCl. We therefore hy- pothesized that DNA helper probes added to a hybrid DNA/PNA system might still perform some level of helper function, with higher concentrations of helper probe being expected to drive the reaction toward completion, compensating for nonoptimal reac- tion conditions. Additionally, a higher NaCl concentration of 300 FIG 4 Specificity and discriminatory power of the P-CalB2208 and P-Ca726 probes examined against a panel comprised of C. albicans and nontarget yeasts. (A) Intensity of FISH results for each test strain, quantified by FCM. (B) Results for analysis of probe discriminatory power, expressed as the ratio of C. albicans ATCC 10231 fluorescence to that of each test strain. Higher values for this ratio indicate each probe’s capacity to discriminate between target and nontarget organisms.
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PNA Biosensors for Nucleic Acid Detection

PNA Biosensors for Nucleic Acid Detection

The immobilization step should result in a readily accessible and yet highly stable PNA probe. While the use of PNA allows the use of very short probes, statistical considerations favor the use of probes longer than 20-mer. Depending upon the nature of the physical transducer, various schemes can be used for attaching the PNA probe to the surface. These include the use of thiolated PNA for self assembly onto gold transducers (gold electrodes or gold-coated piezoelectric crystals), covalent linkage to the gold surface via a functional alkanethiol–based monolayers, the use of biotylated PNA for complex formation with a surface-confined avidin or strepavidin, covalent (carbodiimide) coupling to functional groups on carbon electrodes, or a simple adsorption onto carbon surfaces. The construction of high- density arrays of PNA probes would require the use of microjet deposition technology, involving the dispension of picoliter volumes onto discrete locations on the chip.
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Molecular Beacons of Xeno-Nucleic Acid for Detecting Nucleic Acid

Molecular Beacons of Xeno-Nucleic Acid for Detecting Nucleic Acid

Considering the chemical and biological stabil- ity, PNAs could be used to design gene therapeutic drugs [48,49,50] and other molecular biology and functional genomics [51,52]. Besides, PNA probes are extremely useful in situ hybridization and provide very good chromosome images [53,54,55,56,57,58]. A few early reports have examined the properties of PNAs as both specific [59,60,61] as well as general [62] nucleic acid capture probes. Recently, PNA-based MBs, optoelectronic [63], microarray or electrochem- ical probes [64] have been developed for different biochemical and biotechnological applications [65]. Fang et al. developed an electrochemical nucleic acids probe made of PNA which exhibits high sensitivity and specificity when challenged with heterogeneous samples of RNA. They further used the probes to de- tect a newly identified cancer biomarker-a gene fusion associated with prostate cancer. The system could detect specific mRNAs in unamplified patient sam- ples in as little as 10 ng [66]. PNA was also success- fully used to label-free DNA/PNA hybridization de- tection when combined with silicon-based platform [67].
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Dinuclear polypyridyl ruthenium(II) complexes as stereoselective probes of nucleic acid secondary structures

Dinuclear polypyridyl ruthenium(II) complexes as stereoselective probes of nucleic acid secondary structures

The most conspicuous means by which selectivity might be obtained is to target a specific base sequence. Such a sequence might belong to a specific gene or its promoter or enhancer regions, with the subsequent binding resulting in some degree of modulation of the gene itself. Selectively binding a unique DNA sequence in the human genome would require an incredibly complex drug able to recognise a sequence of some 15-16 bases † . 407-409 Molecules possessed of such a binding footprint present a significant synthetic challenge and potential candidates have, to date, have had limited success. 407, 409 Even so, a smaller target sequence of 6-10 bases (for example) would still occur relatively infrequently within the genome and therefore still represent a viable objective. The lower end of this range corresponds to the footprint size of some of the more specific nucleic acid binders in use today. These ligands are typically polyamide minor groove binders such as netropsin and derivatives thereof. Selectivity is achieved via specific hydrogen-bonding patterns between amide functionalities on the drug molecules and polar minor groove atoms on the target bases. Species featuring pyrrole subunits favourably bind A•T and T•A base pairs, whereas those with imidazole subunits preferably bind to G•C and C•G base pairs. 410-412 Unfortunately, a mismatch in the geometries between the DNA minor groove and polyamide geometries means that the hydrogen-bonding functionalities on each eventually become out of phase (within approximately 10 base pairs). Multiple
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Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ⴝ 72), C. dubliniensis (n ⴝ 58), C. glabrata (n ⴝ 5), C. krusei (n ⴝ 2), C. parapsilosis (n ⴝ 4), and C. tropicalis (n ⴝ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.
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Novel Fatty Acid Desaturases And Elongases And Uses Thereof (Patent WO 2011/006948 A1)

Novel Fatty Acid Desaturases And Elongases And Uses Thereof (Patent WO 2011/006948 A1)

of the nucleic acid molecule, preferably, into a translatable mRNA. Additional regulatory elements may include transcriptional as well as translational enhancers. The following promoters and expression control sequences may be, preferably, used in an expression vector according to the present invention. The cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ -PR or λ -PL promoters are, preferably, used in Gram-negative bacteria. For Gram-positive bacteria, promoters amy and SPO2 may be used. From yeast or fungal promoters ADC1 , AOX1 r, GAL1 , MFa , AC, P-60, CYC1 , GAPDH, TEF, rp28, ADH are, preferably, used. For animal cell or organism expression, the promoters CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40- enhancer are preferably used. From plants the promoters CaMV/35S (Franck 1980, Cell 2 1 : 285-294], PRP1 (Ward 1993, Plant. MoI. Biol. 22), SSU, OCS, Iib4, usp, STLS1 , B33, nos or the ubiquitin or phaseolin promoter. Also preferred in this context are inducible
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How To Make A Nucleic Acid

How To Make A Nucleic Acid

The syntheses of the constructs 1 and 2 were performed fully automatically. Two different resins with two different antisense sequences were chosen to verify the versatility of the presented protocol. More importantly, both the syntheses were performed in parallel which saves time and can be useful in generating PNA libraries easily. The synthesis was started on Wang or Rink amide resins which were downloaded in manual mode in order to avoid aggregation. Construct 3 was synthesized to demonstrate the semi-automated synthesis of a PNA-peptide conjugate. This synthesis was started on Fmoc- D -Arg(Pbf)-Wang resin, downloaded by adding the first amino acid of the sequence (Lys) in the manual mode. The remaining amino acid building blocks and the linker were assembled in the fully automatic mode. Afterwards, the synthesis was continued with the coupling of the PNA sequence in the semi-automated mode. After the assembly of all PNA monomers, the remaining specific building blocks required for our study [e.g. DOTA tris(tert-butyl), FITC] were added manually. After the complete synthesis, the products were cleaved from the resin, precipitated in diethyl ether, purified by HPLC, and molecular masses were confirmed by ESI-MS.
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An Introduction to Peptide Nucleic Acid

An Introduction to Peptide Nucleic Acid

The lack of electrostatic repulsion between the two strands in a PNA/nucleic acid duplex leaves the T m largely independent of salt concentration (Figure 3) (8,10). This allows binding, in the absence of salt, of a PNA probe to a nucleic acid target despite the presence of competing secondary structure (11). It is noticed from the curve that there is a significant difference at physiological salt, and that the T m s are essentially the same at sodium ion concentrations higher than 500 millimolar. The T m for PNA-DNA duplexes does not follow the same rules as for DNA/DNA duplexes. Although the T m correlates with the G-C content it is also highly dependent on the purine content of the PNA strand. In fact, a very useful empirical T m formula based on the T m predicted for the corresponding DNA/DNA duplex, and using correction factors for the purine content of the PNA and the length has been derived (12):
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Polysaccharides as initiators of nucleic acid polymerization

Polysaccharides as initiators of nucleic acid polymerization

A single-stranded DNA was found to form helical structure with a polysaccharide fragment. The characte- ristic of such structure is identical to a double-stranded DNA. The analysis of the interaction between nucleic acids and oligosaccharides carried out alongside the study of the biosynthesis processes of the latter in vivo [2] has enabled us to suggest a concept of template-based synthesis of oligo and polysaccharide molecules with the participation of DNA tandem repeats (glycotranscription process) [3]. However, a question still remains undeter- mined whether DNA formation is possible on one-di- mensional oligosaccharide molecule according to the reverse glycotranscription principle in the way that it was described for RNA-DNA tandem in modern classical molecular biology (the reverse transcription process).
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Characterization of Nucleic Acid of Pichinde Virus

Characterization of Nucleic Acid of Pichinde Virus

Since part of the RNA isolated from Pichinde virions consisted of 28S and 18S components, the possibility that these species were of host cell ribosomal RNA origin was examined by using [r]

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Virion nucleic acid of Ebola virus.

Virion nucleic acid of Ebola virus.

At the outset of this project, no data were available on the nature of the Ebola virus genome, and our analysis considers whether i the virion nucleic acid is RNA or DNA, ii the genome i[r]

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Search for a Prion-Specific Nucleic Acid

Search for a Prion-Specific Nucleic Acid

Studies of synthetic prions combined with investigations of naturally occurring strains support the thesis that prions are devoid of a prion-specific nucleic acid. A novel strain of prions was produced using mouse (Mo) recombinant PrP composed of residues 89 to 230 (36). The first synthetic prion strain (MoSP1) was inoculated into transgenic 9949 mice expressing N-terminally truncated MoPrP( ⌬ 23–88) and wild-type FVB mice expressing full-length MoPrP. Incubation time measure- ments, neuropathologic lesion profiles, and conformational stability studies using guanidinium HCl denaturation indicate that MoSP1 prions differ from RML and many other prion strains derived from humans with Creutzfeldt-Jakob disease, sheep with scrapie, and cattle with bovine spongiform enceph- alopathy (37).
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Algorithms for nucleic acid sequence design

Algorithms for nucleic acid sequence design

By exploiting pairing specificity, one can rationally design sequences of strands such that hybridization energies will drive programmed self-assembly of prescribed molecular structures [3]. This has produced a wide array of engineered nucleic acid systems [4–7] including self-assembling two- and three-dimensional structures, triggered self-assembly mechanisms, computational devices, machines, scaffolds, and catalysts. Despite the different approaches and applications of all these nucleic acid systems, they have an important commonality: they all require the selection of specific sequences that encode the desired structure and func- tion into the system. We refer to this selection process as sequence design.
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Peptide Nucleic Acid Microarrays

Peptide Nucleic Acid Microarrays

A fast and economical procedure for the production of peptide nucleic acid (PNA) microarrays is presented. PNA oligomers are synthesized in a fully automatic manner in 96-well plates using standard Fmoc chemistry. Subsequently, the oligomers are released from the support and spotted onto glass or silicone slides, which were activated by succinimidyl ester. This process allows for a concomitant purification of the oligomers directly on the chip surface. Although the terminal primary amino groups of the full-length products bind selec- tively to this surface, none of the byproducts of synthesis, such as truncated sequences or cleaved side chain protection groups, will bind and are therefore washed away. In this chapter, protocols are presented for the whole production process as well as sample hybridization.
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Freeman Chapter 4 Nucleic Acid Presentation

Freeman Chapter 4 Nucleic Acid Presentation

(1) Antiparallel DNA strands form a double helix—hydrophilic sugar-phosphate backbone faces the exterior, and purine– pyrimidine pairs of nitrogenous bases face the interior.. (2) DNA s[r]

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A Scoring Method for the Clustering of Nucleic Acid Sequences

A Scoring Method for the Clustering of Nucleic Acid Sequences

The clustering of biological sequence data i s a significant task for biologists. The reason is that sequence clustering assists molecular biologists to group sequences based on the ancestral traits or hereditary information that are hidden in sequences. To accomplish the similarity detection and clustering tasks, several clustering algorithms, similarity and d i s t an c e measures have b e e n proposed. Most of these algorithms a n d si milarity measures manifest some form of inefficiency in the detection of sequences based on their structural similarity as was observed in the cou r s e of this study. In this paper, the codon -based scoring method (COBASM) is developed to handle this inefficiency. COBASM employs the codon principle, by the application of triplet nucleotides, in the clustering of nucleic acid sequences. The results obtained show that COBASM is able to produce compact and well- separated clusters based on the structural similarity of sequences.
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Advances in single molecule nucleic acid sequencing

Advances in single molecule nucleic acid sequencing

The sequencing chamber consists of a flexible plastic adhesive flow cell, less than a millimeter in height, which has been applied on the top of a low-autofluorescence microscope coverslip. The sequencing chamber holds approximately 80 µL of fluid. Template DNA is anchored to the surface via the strong binding properties of biotin and streptavidin. Biotin, a modified version containing an amine group, is first covalently bonded to the carboxyl groups of polyacrylic acid, the negatively charged polyelectrolyte, using the catalyst EDC. Next, streptavidin is bound to biotin on the coverslip surface. Template DNA, which has been biotinylated at its 5′ end, is finally applied onto the streptavidin. Thus, streptavidin acts as a bridge between the surface biotin and the biotin of the template DNA. The template DNA has previously been annealed to a Cy3-labeled primer.
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Analysis of the nucleic acid components in reticuloendotheliosis virus.

Analysis of the nucleic acid components in reticuloendotheliosis virus.

Other viruses used were Rauscher leukemia virus in the JLSV-9 BALB/c bone marrow cell line; the endogenous baboon virus M7 isolate grown in a human osteosarcoma line; rat type C virus gr[r]

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Analysis, Design, and Construction of Nucleic Acid Devices

Analysis, Design, and Construction of Nucleic Acid Devices

Given the previously developed tools for the analysis and design of nucleic acid secondary structures, the challenge is to parlay these techniques into useful DNA systems. To this end, we must first conceive of a system of DNA structures predicted to have experimentally interesting behavior, then design sequences to instantiate this framework. Along these lines, we introduce the concept of hybridization chain reaction (HCR), in which stable DNA monomers assemble only upon exposure to a target DNA fragment. In the simplest version of this process, two stable species of DNA hairpins coexist in solution until the introduction of initiator strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. The average molecular weight of the HCR products varies inversely with initiator concentration. Amplification of more diverse recognition events can be achieved by coupling HCR to aptamer triggers. This functionality allows DNA to act as an amplifying transducer for biosensing applications.
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