PubMed Health⌕ Search

Biomedical subjects

Bruce A Armitage

Publications and source records attributed to Bruce A Armitage.

16 recordsLinked to original sources
PubMed2006

Fluorescent PNA probes as hybridization labels for biological RNA.

Fluorescent labeling of biological RNA is complicated by the narrow range of nucleoside triphosphates that can be used for biological synthesis (i.e., transcription) as well as the inability to site-specifically incorporate them into long RNA transcripts. Noncovalent strategies for labeling RNA rely on attaching fluorescent dyes to hybridization probes which deliver the dye to a specific region of the RNA through Watson-Crick base pairing. This report demonstrates the use of high-affinity peptide nucleic acid (PNA) probes in labeling mRNA transcripts with thiazole orange donor and Alexa-594 acceptor fluorophores. The PNA probes were targeted to sequences flanking splice sites in a pre-mRNA such that before splicing the PNAs were separated by >300 nucleotides (nts) whereas after splicing the separation decreased to <or=12 nts. The decreased separation led to enhanced Förster resonance energy transfer (FRET) for the spliced RNA. Bulk solution and single-molecule fluorescence experiments gave consistent results.

Base Sequence↗
PubMed2006

Hybridization of complementary and homologous peptide nucleic acid oligomers to a guanine quadruplex-forming RNA.

Peptide nucleic acid (PNA) oligomers targeted to guanine quadruplex-forming RNAs can be designed in two different ways. First, complementary cytosine-rich PNAs can hybridize by the formation of Watson-Crick base pairs, resulting in hybrid PNA-RNA duplexes. Second, guanine-rich homologous PNAs can hybridize by the formation of G tetrads, resulting in hybrid PNA-RNA quadruplexes. UV thermal denaturation, circular dichroism, and fluorescence spectroscopy experiments were used to compare these two recognition modes and revealed 1:1 duplex formation for the complementary PNA and 2:1 (PNA2-RNA) quadruplex formation for the homologous PNA. Both hybrids were very stable, and hybridization was observed at low nanomolar concentrations. Hybrid quadruplex formation was equally efficient regardless of the PNA strand polarity, indicating a lack of interaction between the loop nucleobases on the PNA and RNA strands. The implications of this finding on sequence specificity as well as methods to improve affinity are also discussed.

Animals↗
PubMed2005

1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove.

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. 1H NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.

Benzothiazoles↗
PubMed2005

RNA guanine quadruplex invasion by complementary and homologous PNA probes.

Guanine quadruplexes are gaining increasing attention due to their suspected roles in regulating gene expression at the transcriptional and translational levels. This paper describes the ability of short peptide nucleic acid (PNA) probes to disrupt a stable RNA quadruplex and hybridize to their target sequence. In one case, the PNA probe is complementary to the target, resulting in formation of a Watson-Crick base-paired duplex. In the second case, the PNA probe is homologous to the target and forms a hybrid quadruplex structure. The hybrid duplex is formed in a 1:1 stoichiometry, as expected based on the constraints imposed by Watson-Crick pairing. However, the hybrid quadruplex is formed in a PNA2:RNA stoichiometry, due to the ability of the short PNA to hybridize with both halves of the original RNA quadruplex.

Base Sequence↗
PubMed2005

Role reversal in a supramolecular assembly: a chiral cyanine dye controls the helicity of a peptide-nucleic acid duplex.

A new dicarbocyanine dye bearing branched, chiral N-alkyl substituents was synthesized and its ability to form helical aggregates on peptide nucleic acid (PNA) double-helical templates was studied. The dye aggregates less effectively than an analogous dye bearing linear, achiral substituents, presumably due to steric problems with packing the branched substituents compared with the linear substituents. When the PNA duplex has a left-handed helicity, addition of the achiral dye leads to formation of a left-handed dye aggregate. However, when the chiral dye aggregates in the presence of this duplex, a right-handed structure is formed, suggesting that the dye alters the helicity of the underlying template. When a racemic PNA duplex (i.e., equal amounts of right- and left-handed helices) is used, no chirality is observed for the dye aggregate formed by the achiral dye but a right-handed helical aggregate is once again formed by the chiral dye. These results indicate that chirality is transferred from the dye to the PNA, as opposed to other examples of polymer-templated dye aggregation where chirality is transferred from the template to the dye.

Carbocyanines↗
PubMed2005

Quadruplex formation by a guanine-rich PNA oligomer.

A guanine-rich PNA dodecamer having the sequence H-G4T4G4-Lys-NH2 (G-PNA) hybridizes with a DNA dodecamer of homologous sequence to form a four-stranded quadruplex (Datta, B.; Schmitt, C.; Armitage, B. A. J. Am. Chem. Soc. 2003, 125, 4111-4118). This report describes quadruplex formation by the PNA alone. UV melting curves and fluorescence resonance energy transfer experiments reveal formation of a multistranded structure stabilized by guanine tetrads. The ion dependency of these structures is analogous to that reported for DNA quadruplexes. Electrospray ionization mass spectrometry indicates that both dimeric and tetrameric quadruplexes are formed by G4-PNA, with the dimeric form being preferred. These results have implications for the use of G-rich PNA for homologous hybridization to G-rich targets in chromosomal DNA and suggest additional applications in assembling quadruplex structures within lipid bilayer environments.

Base Sequence↗
PubMed2005

Effect of PNA backbone modifications on cyanine dye binding to PNA-DNA duplexes investigated by optical spectroscopy and molecular dynamics simulations.

Optical spectroscopy and molecular dynamics simulations have been used to study the interaction between a cationic cyanine dye and peptide nucleic acid (PNA)-DNA duplexes. This recognition event is important because it leads to a visible color change, signaling successful hybridization of PNA with a complementary DNA strand. We previously proposed that the dye recognized the minor groove of the duplex, using it as a template for the assembly of a helical aggregate. Consistent with this, we now report that addition of isobutyl groups to the PNA backbone hinders aggregation of the dye when the substituents project into the minor groove but have a weaker effect if directed out of the groove. UV-Visible and circular dichroic spectroscopy were used to compare aggregation on the different PNA-DNA duplexes, while molecular dynamics simulations were used to confirm that the substituents block the minor groove to varying degrees, depending on the configuration of the starting amino acid. In addition to a simple steric blockage effect of the substituent, the simulations suggest that directing the isobutyl group into the minor groove causes the groove to narrow and the duplex to become more rigid, structural perturbations that are relevant to the growing interest in backbone-modified PNA for applications in the biological and materials sciences.

Benzothiazoles↗
PubMed2004

Improved photostability and fluorescence properties through polyfluorination of a cyanine dye.

[structure: see text] A polyfluorinated cyanine dye has been synthesized and characterized. Compared with the nonfluorinated analogue, the dye exhibits significantly reduced aggregation in aqueous media, enhanced fluorescence quantum yield, greater resistance to photobleaching upon direct irradiation, and reduced reactivity toward singlet oxygen. All of these properties are favorable for use of cyanine dyes as fluorescent labels and point toward fluorination as a general strategy for improving performance in imaging applications.

Carbocyanines↗
PubMed2004

A recoverable enzymatic microgel based on biomolecular recognition.

The enzyme beta-galactosidase has been immobilized through incorporation into a selectively soluble microgel, prepared from DNA, biotinylated peptide nucleic acid (PNA), and the protein avidin. The enzyme was conjugated to avidin, allowing it to be integrated directly into the microgel network. Efficient hydrolysis of a small-molecule substrate occurred at 37 degrees , but cooling and centrifuging led to precipitation of the microgels and product separation. The microgels were then reconstituted by adding fresh buffer and shaking. The enzymatic activity was completely recovered through repeated cycles. This method should be generalizable to a wide variety of other enzymes and substrates.

Avidin↗
PubMed2004

DNA-templated assembly of helical cyanine dye aggregates: a supramolecular chain polymerization.

Symmetrical cationic cyanine dyes assemble in cooperative fashion into helical supramolecular polymers using DNA as a template. The dyes assemble into cofacial dimers within the minor groove of the DNA and assembly of one dimer facilitates assembly of additional dimers directly adjacent to the first. Growth of the polymer ceases when the end of the DNA is reached or when the DNA sequence blocks dimerization of the dye. Thus, this process can be thought of as a supramolecular analogue of a chain polymerization. This Account describes how polymerization depends on the dye structure and DNA sequence and also summarizes the interesting optical properties exhibited by these chiral, helical materials.

Base Sequence↗
PubMed2004

Recent advances in the development of peptide nucleic acid as a gene-targeted drug.

Peptide nucleic acid (PNA) is a non-ionic mimic of DNA that binds to complementary DNA and RNA sequences with high affinity and selectivity. Targeting of single-stranded RNA leads to antisense effects, whereas PNAs directed toward double-stranded DNA exhibit antigene properties. Recent advances in cell uptake and in antisense and antigene effects in biological systems are summarised in this review. In addition to traditional targets, namely genomic DNA and messenger RNA, applications for PNA as a bacteriocidal antibiotic, for regulating splice site selection and as a telomerase inhibitor are described.

Animals↗
PubMed2003

Synthesis and characterization of thermoreversible biopolymer microgels based on hydrogen bonded nucleobase pairing.

We describe the synthesis and characterization of a thermoreversibly cross-linked biopolymer microgel based on protein, DNA, and peptide nucleic acid (PNA) components. The DNA assembles into a trifunctional three-way junction (TWJ) with single-stranded overhangs. PNA oligomers complementary to these overhangs and bearing terminal biotin groups hybridize to the DNA TWJ and simultaneously bind to the tetrafunctional protein avidin, leading to a cross-linked system. Dynamic light scattering experiments reveal that micron-sized particles are formed. Static light scattering was used to characterize the internal structure of these microgels, which were found to have a fractal dimension of 1.85, indicative of a loose network structure. Heating disrupts the weakest component in the system, namely the PNA-DNA hybrid, resulting in dissolution of the microgel, while cooling restores the hydrogen bonding leading to reassembly of the microgel. Variation of the nucleotide sequence permits tuning of the gelation temperature with fine control.

Avidin↗
PubMed2003

Formation of a PNA2-DNA2 hybrid quadruplex.

DNA guanine (G) quadruplexes are stabilized by an interesting variation of the hydrogen-bonding schemes encountered in nucleic acid duplexes and triplexes. In an attempt to use this mode of molecular recognition, we target a dimeric G-quadruplex formed by the Oxytricha nova telomeric sequence d(G(4)T(4)G(4)) with a peptide nucleic acid (PNA) probe having a homologous rather than complementary sequence. UV-vis and CD spectroscopy reveal that a stable hybrid possessing G-quartets is formed between the PNA and DNA. The four-stranded character of the hybrid and the relative orientation of the strands is determined by fluorescence resonance energy transfer (FRET) experiments. FRET results indicate that (i) the two PNA strands are parallel to each other, (ii) the two DNA strands are parallel to each other, and (iii) the 5'-termini of the DNA strands align with the N-termini of the PNA strands. The resulting PNA(2)-DNA(2) quadruplex shows a preference of Na(+) over Li(+) and displays thermodynamic behavior consistent with alternating PNA and DNA strands in the hybrid. The formation of this novel supramolecular structure demonstrates a new high-affinity DNA recognition mechanism and expands the scope of molecular recognition by PNA.

Animals↗
PubMed2003

The impact of nucleic acid secondary structure on PNA hybridization.

Hybridization of oligonucleotides and their analogues to complementary DNA or RNA sequences is complicated by the presence of secondary and tertiary structure in the target. In particular, folding of the target nucleic acid imposes substantial thermodynamic penalties to hybridization. Slower kinetics for hybridization can also be observed, relative to an unstructured target. The development of high affinity oligonucleotide analogues such as peptide nucleic acid (PNA) can compensate for the thermodynamic and kinetic barriers to hybridization. Examples of structured targets successfully hybridized by PNA oligomers include DNA duplexes, DNA hairpins, DNA quadruplexes and an RNA hairpin embedded within a mRNA.

Nucleic Acid Hybridization↗