A new family of sequence-specific DNA-cleaving agents directed by triple-helical structures: benzopyridoindole-EDTA conjugates

Sequence-specific DNA recognition can be achieved by oligonucleotides that bind to the major groove of oligopyrimidine x oligopurine sequences. These intermolecular structures could be used to modulate gene expression and to create new tools for molecular biology. Here we report the synthesis and biochemical characterization of triple helix-specific DNA cleaving reagents. It is based on the previously reported triplex-specific ligands, benzo[e]pyridoindole (BePI) and benzo[g]pyridoindole (BgPI), covalently attached to ethylenediaminotetraacetic acid (EDTA). In the presence of iron, a reducing agent and molecular oxygen, BgPI-EDTA x FeII but not BePI-EDTA x FeII induced a double-stranded cut in a plasmid DNA at the single site where a triplex-forming oligonucleotide binds. At single nucleotide resolution, it was found that upon triplex formation BePI-EDTA x FeII led to cleavage of the pyrimidine strand and protection of the purine strand. BgPI-EDTA x FeII cleaved both strands with similar efficiency. The difference in cleavage efficiency between the two conjugates was rationalized by the location of the EDTA x FeII moiety with respect to the grooves of DNA (major groove: BePI-EDTA x FeII, minor groove: BgPI-EDTA x FeII). This work paves the way to the development of a new class of triple helix directed DNA cleaving reagents. Such molecules will be of interest for sequence-specific DNA cleavage and for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.     

Forest Biophysical Parameter Estimation Using L- and P-Band Polarimetric SAR Data

L- and P-band airborne polarimetric synthetic aperture radar (SAR) data acquired by the RAMSES system over different height maritime pine (Pinus Pinaster Ait.) stands of the Nezer forest (Landes, France) have been evaluated for forest biophysical parameter estimation. A pseudolinear correlation has been brought to evidence at P-band between polarimetric anisotropy and mean tree height, which is also linked to other biophysical parameters in the Nezer forest, meaning that SAR polarimetry constitutes a promising tool for forest parameter retrieval at low frequency. The spatial conditions have been evaluated through the quantification of the impact of signal-to-noise ratio diminution and resolution degradation on the forest height inversion. It has been shown that the inversion accuracy remains acceptable for N Esigma₀, representing the noise level of the SAR image, which is lower than -15 dB, and for spatial resolution increasing up to 15 m.     

Directing Topoisomerase I Mediated DNA Cleavage to Specific Sites by Camptothecin Tethered to Minor- and Major-Groove Ligands

No Abstract     

Bridging the gap between proteins and nucleic acids: a metal-independent RNAseA mimic with two protein-like functionalities.

Two synthetically modified nucleoside triphosphate analogues (adenosine modified with an imidazole and uridine modified with a cationic amine) are enzymatically polymerized in tandem along a degenerate DNA library for the combinatorial selection of an RNAse A mimic. The selected activity is consistent with both electrostatic and general acid/base catalysis at physiological pH in the absence of divalent metal cations. The simultaneous use of two modified nucleotides to enrich the catalytic repertoire of DNA-based catalysts has never before been demonstrated and evidence of general acid/base catalysis at pH 7.4 for a DNAzyme has never been previously observed in the absence of a divalent metal cation or added cofactor. This work illustrates how the incorporation of protein-like functionalities in nucleic acids can bridge the gap between proteins and oligonucleotides underscoring the potential for using nucleic acid scaffolds in the development of new materials and improved catalysts for use in chemistry and medicine.     

Pine Forest Height Inversion Using Single-Pass X-Band PolInSAR Data

A sparse pine forest is investigated at X-band on a single-pass polarimetric synthetic aperture radar interferometry (PolInSAR) data set using HH and HV channels. These first preliminary results show that the associated phase centers present a significant vertical separation (about 6 m) allowed by penetration through gaps in the canopy. Forest parameter inversion using the random volume over ground (RVoG) model is evaluated and adapted at this frequency. The forest height can be retrieved accurately by supposing a high mean extinction coefficient (around 1.6 (dB/m). The penetration depth is estimated to be around 4 m, based on the forest height ground measurements. Finally, a time-frequency analysis using a sublook decomposition is performed to increase the vertical separation of the polarimetric phase centers. As a consequence, RVoG-inversion performance is improved, and a penetration depth that is in better accordance with a previous work (of the order of 2 m) is found. This paper has shown that the height inversion of a pine forest was possible using PolInSAR X-band data and that the performance was more dependent on the forest density than at lower frequencies.     

Design of a triple-helix-specific cleaving reagent

BACKGROUND: Double-helical DNA can be recognized sequence specifically by oligonucleotides that bind in the major groove, forming a local triple helix. Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive study. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We previously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) can strongly stabilize triple helices. RESULTS: We have designed and synthesized the first triple-helix-specific DNA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacetic acid (EDTA). The intercalative binding of BQQ should position EDTA in the minor groove of the triple helix. In the presence of Fe(2+) and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) DNA fragment at the site where an oligonucleotide binds to form a local triple helix. The selectivity of the BQQ-EDTA conjugate for a triplex structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA. CONCLUSIONS: This new class of structure-directed DNA cleaving reagents could be useful for cleaving DNA at specific sequences in the presence of a site-specific, triple-helix-forming oligonucleotide and also for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.     

New junction models for alternate-strand triple-helix formation

Background: Oligonucleotide-directed triple-helix (triplex) formation can interfere with gene expression but only long tracts of oligopyrimidine·oligopurine sequences can be targeted. Attempts have been made to recognize short oligopurine sequences alternating on the two strands of double-stranded DNA by the covalent linkage of two triplex-forming oligonucleotides. Here we focus on the rational optimization of such an alternate-strand triplex formation on a DNA duplex containing a 5′-GpT-3′/3′-CpA-5′ or a 5′-TpG-3′/3′-ApC-5′ step by combination of (G,T)- and (G,A)-contain ing oligonucleotides that bind to the oligopurine strands in opposite orientations.Results: The deletion of one nucleotide in the reverse Hoogsteen region of the oligonucleotide provides the best binding at the 5′-GpT-3′/3′-CpA-5′ step, whereas the addition of two cytosines as a linker between the two oligonucleotides is the best strategy to cross a 5′-TpG-3′/3′-ApC-5′ step. Energy minimization and experimental data suggest that these two cytosines are involved in the formation of two novel base quadruplets.Conclusions: These data provide a rational basis for the design of oligonucleotides capable of binding to oligopurine sequences that alternate on the two strands of double-stranded DNA with a 5′-GpT-3′/3′-CpA-5′ or a 5′-TpG-3′/3′-ApC-5' step at the junction.     

PolInSAR analysis of X-band data over vegetated and urban areas

This paper investigates the polarimetric and polarimetric interferometric synthetic aperture radar (PolInSAR) information contained in the high-resolution X-band data acquired by the RAMSES airborne SAR system over an area around Avignon, France containing bare surfaces, vegetation, and urban areas. The interferometric coherences are computed over natural and urban areas for all possible baseline copolar polarizations. In the complex plane, the obtained regions of coherence corresponding to most vegetation areas display small angular extents, meaning that if penetration occurs in the foliage, it is shallower than the system height accuracy. To quantify the PolInSAR information, an analysis of the interferometric height accuracy is first performed, and the results are compared with those associated with a theoretical and an empirical model. Concerning vegetation, a 6-m height difference is measured between the different polarimetric phase centers over a sparse pine forest, probably due to the presence of holes in the canopy. Crop study reveals also that wheat-type fields present oriented media properties at X-band due to their vertical structure. Over urban areas, in most cases, building height can be accurately obtained by using Pauli polarimetric phase center information.     

Direct photocleavage of HIV-DNA by quinacridine derivatives triggered by triplex formation

Amino-p-quinacridine compounds (PQs) have been shown to stabilize strongly and specifically triple-helical DNA. Moreover, these derivatives display photoactive properties that make them efficient DNA cleavage agents. We exploited these two properties (triplex-specific binding and photoactivity) to selectively cleave a double-stranded (ds)DNA sequence present in the HIV-1 genome. Cleavage was first carried out on a linearized plasmid (3300 bp) containing the HIV polypurine tract (PPT) that allowed targeting by a triplex-forming oligonucleotide (TFO). PQ(3)(), the most active compound of the series, efficiently cleaved double-stranded DNA in the vicinity of the PPT when this sequence had formed a triplex with a 16-mer TFO. Investigation of the cleavage at the molecular level was addressed on a short DNA fragment (56 bp); the photoinduced cleavage by PQ(3)() occurred only in the presence of the triple helix. Nevertheless, unusual cleavage patterns were observed: damage was observed at guanines located 6-9 bp away from the end of the triple helical site. This cleavage is very efficient (up to 60%), does not require alkaline treatment, and is observed on both strands. A quinacridine-TFO conjugate produced the same cleavage pattern. This observation, along with others, excludes the hypothesis of a triplex-induced allosteric binding site of PQ(3 )()adjacent to the damaged sequence and indicates that PQ(3 )()preferentially binds in the vicinity of the 5'-triplex junction. Irradiation in the presence of TFO-conjugates with acridine (an intercalative agent) and with the tripeptide lys-tryp-lys led to a complete inhibition of the photocleavage reaction. These results are interpreted in terms of competitive binding and of electron-transfer quenching. Together with the findings of simple mechanistic investigations, they led to the conclusion that the photoinduced damage proceeds through a direct electron transfer between the quinacridine and the guanines. This study addresses the chemical mechanism leading to strand breakage and characterizes the particular photosensitivity of the HIV-DNA target sequence which could be an oxidative hot spot for addressed photoinduced strand scission by photosensitizers.