Ligand-induced formation of triple helices with antiparallel third strands containing G and T

We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple-helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I.     

Rational design of a triple helix-specific intercalating ligand

DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them. Molecular modeling and thermal denaturation experiments suggest that benzo[f]pyrido[3, 4-b]quinoxaline derivatives intercalate into triple helices by stacking preferentially with the Hoogsteen-paired bases. Based on this model, it was predicted that a benzo[f]quino[3,4-b]quinoxaline derivative, which possesses an additional aromatic ring, could engage additional stacking interactions with the pyrimidine strand of the Watson-Crick double helix upon binding of this pentacyclic ligand to a triplex structure. This compound was synthesized. Thermal denaturation experiments and inhibition of restriction enzyme cleavage show that this new compound can indeed stabilize triple helices with great efficiency and specificity and/or induce triple helix formation under physiological conditions.     

Pine forest investigation using high resolution P-band Pol-InSAR data

A Pol-InSAR dataset acquired at P-band over a maritime pine forest (Nezer forest of Landes, France) has been investigated and compared with tree structural ground measurements in order to identify scatterers associated with elementary scattering mechanisms in the canopy layer. Since this managed forest is constituted by different height stands, it allows to investigate mechanism change in the vertical direction during the forest growth. It has been observed that VV channel was the most elevated, which can be accounted for a maximum attenuation in the vertical direction and partially for the presence of sub-vertical branches in the top canopy layer, and that this channel was the most sensitive to the tree density in reason of an important ground reflectivity. The most pure double-bounce scattering mechanism component is received in HH-VV channel where the corresponding phase center reaches the ground level for large Diameter at Breast Height. It is also supposed that HH channel, which receives double bounce backscatter, was affected by the sub-horizontal primary branch development, which induces an elevation of the phase center over oldest trees while tree density diminution enhances backscatter from the ground level. Finally, the crosspolar channel appeared the less perturbed by the tree density than VV channel since it receives predominantly the canopy contribution.     

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.     

Triple helices formed at oligopyrimidine*oligopurine sequences with base pair inversions: effect of a triplex-specific ligand on stability and selectivity

Oligonucleotide-directed triple helix formation is mostly restricted to oligopyrimidine*oligopurine sequences of double helical DNA. An interruption of one or two pyrimidines in the oligopurine target strand leads to a strong triplex destabilisation. We have investigated the effect of nucleotide analogues introduced in the third strand at the site opposite the base pair inversion(s). We show that a 3-nitropyrrole derivative (M) discriminates G*C from C*G, A*T and T*A in the presence of a triplex-specific ligand (a benzo[e]pyridoindole derivative, BePI). N6-methoxy-2,6-diaminopurine (K) binds to an A*T base pair better than a T*A, G*C or C*G base pair. Some discrimination is still observed in the presence of BePI and triplex stability is markedly increased. These findings should help in designing BePI-oligonucleotide conjugates to extend the range of DNA sequences available for triplex formation.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

Triple helix formation by (G,A)-containing oligonucleotides: asymmetric sequence effect

Sequence effects on the stability of purine-motif (also called (G, A)-motif) triple helix have been investigated through two symmetry-related systems: one of them had a 5'(GGA)43' core sequence of triplex-forming oligonucleotides (TFOs), whereas the other one had a reversed 5'(AGG)43' core sequence. These (G,A)-containing TFOs were prone to self-associate into intermolecular complexes at room temperature. The competition of TFOs' self-association with triple helix formation was assessed, and minimized. By varying the lengths and the terminal base sequences of TFOs, the following were found that (1) The stability of two triple helices with identical length and base composition but reverse strand orientation may be significantly different (up to a factor of 6). (2) When the 5'(GGA)43' core sequence was extended at the 3'-end by a G, the 13-nt TFO exhibited 3- and 5-fold higher affinity toward the target double-stranded DNA (dsDNA) than the longer 14-nt and 15-nt TFOs in which one and two A(s) were added at the 3'-end of the 13-nt TFO, respectively. In contrast, when the similar extensions occurred at the 5'-end of the 5'(AGG)43' core sequence, the length increase provided a higher binding affinity of TFOs toward the target duplex. (3) The nature of the base triplets involved at the ends of triple helices may have great influence on triplex stability. The observed asymmetric sequence effect of the (G,A)-motif triple helix formation is discussed in terms of the binding strength of the first base triplet(s) at the 3' end which seems to be deeply involved in the nucleation step of triple helix formation and therefore to be a determining factor for triplex stability.     

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.     

Triple-helix directed cleavage of double-stranded DNA by benzoquinoquinoxaline-1,10-phenanthroline conjugates

Oligonucleotide-directed triple-helix formation provides a rational means to interfere with genomic DNA targets and to direct modifications at specific sites. We have developed a new class of compounds that, at low concentrations, efficiently targets and damages double-stranded DNA specifically at the site where a triple-helical structure is formed. In these new compounds, a triple-helix-specific intercalator-benzoquinoquinoxaline (BQQ)-was coupled to one of two isomeric 1,10-phenanthrolinecarboxaldehyde derivatives. 1,10-Phenanthroline derivatives are known to cleave DNA in the presence of copper ions. The obtained BQQ-1,10-phenanthroline (BQQ-OP) conjugates were compared with regard to their ability to cleave triple-helix DNA. Both conjugates displayed a sequence preference inside the triple-helical site, as judged from the more pronounced cleavage obtained at stretches of TAxT base triplets.     

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.