Binding-driven reactivity attenuation enables NMR identification of selective drug candidates for nucleic acid targets.

NMR methods, and in particular ligand-based approaches, are among the most robust and reliable alternatives for binding detection and consequently, they have become highly popular in the context of hit identification and drug discovery. However, when dealing with DNA/RNA targets, these techniques face limitations that have precluded widespread application in medicinal chemistry. In order to expand the arsenal of spectroscopic tools for binding detection and to overcome the existing difficulties, herein we explore the scope and limitations of a strategy that makes use of a binding indicator previously unexploited by NMR: the perturbation of the ligand reactivity caused by complex formation.

NMR Characterization of Angiogenin Variants and tRNA Products Impacting Aberrant Protein Oligomerization.

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease.

Glucose-nucleobase pairs within DNA: impact of hydrophobicity, alternative linking unit and DNA polymerase nucleotide insertion studies.

Recently, we studied glucose-nucleobase pairs, a binding motif found in aminoglycoside-RNA recognition. DNA duplexes with glucose as a nucleobase were able to hybridize and were selective for purines. They were less stable than natural DNA but still fit well on regular B-DNA.

Mapping the affinity landscape of Thrombin-binding aptamers on 2΄F-ANA/DNA chimeric G-Quadruplex microarrays.

In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2΄-Fluoroarabinonucleic acid (2΄F-ANA) is a prime candidate for such use in microarrays. Indeed, 2΄F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered.

Glucose-Nucleobase Pseudo Base Pairs: Biomolecular Interactions within DNA.

Noncovalent forces rule the interactions between biomolecules. Inspired by a biomolecular interaction found in aminoglycoside-RNA recognition, glucose-nucleobase pairs have been examined. Deoxyoligonucleotides with a 6-deoxyglucose insertion are able to hybridize with their complementary strand, thus exhibiting a preference for purine nucleobases.

Finding the Right Candidate for the Right Position: A Fast NMR-Assisted Combinatorial Method for Optimizing Nucleic Acids Binders.

Development of strong and selective binders from promiscuous lead compounds represents one of the most expensive and time-consuming tasks in drug discovery. We herein present a novel fragment-based combinatorial strategy for the optimization of multivalent polyamine scaffolds as DNA/RNA ligands. Our protocol provides a quick access to a large variety of regioisomer libraries that can be tested for selective recognition by combining microdialysis assays with simple isotope labeling and NMR experiments.

The structural impact of DNA mismatches.

The structure and dynamics of all the transversion and transition mismatches in three different DNA environments have been characterized by molecular dynamics simulations and NMR spectroscopy. We found that the presence of mismatches produced significant local structural alterations, especially in the case of purine transversions. Mismatched pairs often show promiscuous hydrogen bonding patterns, which interchange among each other in the nanosecond time scale.

Effects of sugar functional groups, hydrophobicity, and fluorination on carbohydrate-DNA stacking interactions in water.

Carbohydrate-aromatic interactions are highly relevant for many biological processes. Nevertheless, experimental data in aqueous solution relating structure and energetics for sugar-arene stacking interactions are very scarce. Here, we evaluate how structural variations in a monosaccharide including carboxyl, N-acetyl, fluorine, and methyl groups affect stacking interactions with aromatic DNA bases.

The effect of loop residues in four-stranded dimeric structures stabilized by minor groove tetrads.

Some DNA oligonucleotides can fold back and self-associate forming dimeric structures stabilized by intermolecular base pairs. The resulting antiparallel dimer is a tightly packed four-stranded structure formed by a core of minor groove tetrads connected by short loops of unpaired nucleotides. We have explored the sequential requirements for the loop residues and have found that this family of structures is only stable with one- and two-residue loops, with the stability of the former ones being only marginal.

Carbohydrate-DNA interactions at G-quadruplexes: folding and stability changes by attaching sugars at the 5'-end.

Quadruplex DNA structures are attracting an enormous interest in many areas of chemistry, ranging from chemical biology, supramolecular chemistry to nanoscience. We have prepared carbohydrate-DNA conjugates containing the oligonucleotide sequences of G-quadruplexes (thrombin binding aptamer (TBA) and human telomere (TEL)), measured their thermal stability and studied their structure in solution by using NMR and molecular dynamics. The solution structure of a fucose-TBA conjugate shows stacking interactions between the carbohydrate and the DNA G-tetrad in addition to hydrogen bonding and hydrophobic contacts.

Somatostatin subtype-2 receptor-targeted metal-based anticancer complexes.

Conjugates of a dicarba analogue of octreotide, a potent somatostatin agonist whose receptors are overexpressed on tumor cells, with [PtCl(2)(dap)] (dap = 1-(carboxylic acid)-1,2-diaminoethane) (3), [(η(6)-bip)Os(4-CO(2)-pico)Cl] (bip = biphenyl, pico = picolinate) (4), [(η(6)-p-cym)RuCl(dap)](+) (p-cym = p-cymene) (5), and [(η(6)-p-cym)RuCl(imidazole-CO(2)H)(PPh(3))](+) (6), were synthesized by using a solid-phase approach. Conjugates 3-5 readily underwent hydrolysis and DNA binding, whereas conjugate 6 was inert to ligand substitution. NMR spectroscopy and molecular dynamics calculations showed that conjugate formation does not perturb the overall peptide structure.

Synthesis and structural characterization of stable branched DNA g-quadruplexes using the trebler phosphoramidite.

Guanine (G)-rich sequences can form a noncanonical four-stranded structure known as the G-quadruplex. G-quadruplex structures are interesting because of their potential biological properties and use in nanosciences. Here, we describe a method to prepare highly stable G-quadruplexes by linking four G-rich DNA strands to form a monomolecular G-quadruplex.

Apolar carbohydrates as DNA capping agents.

Mono- and disaccharides have been shown to stack on top of DNA duplexes stabilizing sequences with terminal C-G base pairs. Here we present an apolar version of glucose and cellobiose as new capping agents that stack on DNA increasing considerably its stability with respect to their natural polyhydroxylated mono- and disaccharide DNA conjugates.

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly.

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I.

Identification of ligands for the Tau exon 10 splicing regulatory element RNA by using dynamic combinatorial chemistry.

We describe the use of dynamic combinatorial chemistry (DCC) to identify ligands for the stem-loop structure located at the exon 10-5'-intron junction of Tau pre-mRNA, which is involved in the onset of several tauopathies including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). A series of ligands that combine the small aminoglycoside neamine and heteroaromatic moieties (azaquinolone and two acridines) have been identified by using DCC. These compounds effectively bind the stem-loop RNA target (the concentration required for 50% RNA response (EC(50)): 2-58 μM), as determined by fluorescence titration experiments.

Highly polar carbohydrates stack onto DNA duplexes via CH/π interactions.

Carbohydrate-nucleic acid contacts are known to be a fundamental part of some drug-DNA recognition processes. Most of these interactions occur through the minor groove of DNA, such as in the calicheamicin or anthracycline families, or through both minor and major groove binders such as in the pluramycins. Here, we demonstrate that carbohydrate-DNA interactions are also possible through sugar capping of a DNA double helix.

Differential stability of 2'F-ANA*RNA and ANA*RNA hybrid duplexes: roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility.

Hybrids of RNA with arabinonucleic acids 2'F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2'F-ANA*RNA and ANA*RNA hybrids indicates that the increased binding affinity of 2'F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2'F-purine H8) which contrasts with unfavorable 2'-OH-nucleobase steric interactions in the case of ANA.

Self-association of short DNA loops through minor groove C:G:G:C tetrads.

In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution.

Discovery of ligands for a novel target, the human telomerase RNA, based on flexible-target virtual screening and NMR.

The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1.

Four-stranded DNA structures can be stabilized by two different types of minor groove G:C:G:C tetrads.

Four-stranded nucleic acid structures are central to many processes in biology and in supramolecular chemistry. It has been shown recently that four-stranded DNA structures are not only limited to the classical guanine quadruplex but also can be formed by tetrads resulting from the association of Watson-Crick base pairs. Such an association may occur through the minor or the major groove side of the base pairs.

Synthesis and testing of a focused phenothiazine library for binding to HIV-1 TAR RNA.

We have synthesized a series of phenothiazine derivatives, which were used to test the structure-activity relationship of binding to HIV-1 TAR RNA. Variations from our initial compound, 2-acetylphenothiazine, focused on two moieties: ring substitutions and n-alkyl substitutions. Binding characteristics were ascertained via NMR, principally by saturation transfer difference spectra of the ligand and imino proton resonance shifts of the RNA.

Induced-fit recognition of DNA by small circular oligonucleotides.

We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d with d, as well as with its two linear analogs d(GTCCCTCA) and d(CTCAGTCC).

Effect of bulky lesions on DNA: solution structure of a DNA duplex containing a cholesterol adduct.

The three-dimensional solution structure of two DNA decamers of sequence d(CCACXGGAAC)-(GTTCCGGTGG) with a modified nucleotide containing a cholesterol derivative (X) in its C1 '(chol)alpha or C1 '(chol)beta diastereoisomer form has been determined by using NMR and restrained molecular dynamics. This DNA derivative is recognized with high efficiency by the UvrB protein, which is part of the bacterial nucleotide excision repair, and the alpha anomer is repaired more efficiently than the beta one. The structures of the two decamers have been determined from accurate distance constraints obtained from a complete relaxation matrix analysis of the NOE intensities and torsion angle constraints derived from J-coupling constants.