Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

New triple-helix DNA stabilizing agents

Several substituted quinolin-4-amines and heteroaromatic analogs were synthesized and evaluated for interaction with triplex polydA.2polydT and duplex polydA.polydT by using UV-thermal melting experiments. Excellent triple-helix DNA ligands with high affinity toward T.A.T triplets and triple/duplex selectivity were designed through a rational approach.     

DNA purification by triple-helix affinity precipitation

Recent advances in DNA-based medicine (gene therapy, genetic vaccination) have intensified the necessity for pharmaceutical-grade plasmid DNA purification at comparatively large scales. In this contribution triple-helix affinity precipitation is introduced for this purpose. A short, single-stranded oligonucleotide sequence (namely (CTT)(7)), which is capable of recognizing a complementary sequence in the double-stranded target (plasmid) DNA, is linked to a thermoresponsive N-isopropylacrylamide oligomer to form a so-called affinity macroligand (AML). At 4 degrees C, i.e., below its critical solution temperature, the AML binds specifically to the target molecule in solution; by raising the temperature to 40 degrees C, i.e., beyond the critical solution temperature of the AML, the complex can be precipitated quantitatively. After redissolution of the complex at lower temperature, the target DNA can be released by a pH shift to slightly alkaline conditions (pH 9.0). Yields of highly pure (plasmid) DNA were routinely between 70% and 90%. Non-specific co- precipitation of either the target molecule by the non-activated AML precursor or of contaminants by the AML were below 7% and presumably due to physical entrapment of these molecules in the wet precipitate. Ligand efficiencies were at least 1 order of magnitude higher than in triple-helix affinity chromatography.     

DNA triple-helix formation at pyrimidine-purine inversion sites

A systematic investigation of a series of triplex forming oligonucleotides (TFOs) containing alpha-, and beta-thymidine, alpha- and beta-N7-hypoxanthine, and alpha- and beta-N7 and N9 aminopurine nucleosides, designed to bind to T-A inversion sites in DNA target sequences was performed. Data obtained from gel mobility assays indicate that T-A recognition in the antiparallel triple-helical binding motif is possible if the nucleoside alpha N9-aminopurine is used opposite to the inversion site in the TFO.     

Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA

Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k2obs, is 1.8 x 10(3) +/- 1.9 x 10(2) L.(mol of oligomer-1.s-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.     

DNA triple-helix formation at target sites containing duplex mismatches

We have studied the formation of DNA triple helices at target sites that contain mismatches in the duplex target. Fluorescence melting studies were used to examine a series of parallel triple helices that contain all 64 N.XZ triplet combinations at the centre (where N, X and Z are each of the four natural DNA bases in turn). Similar experiments were also performed with N=bis-amino-U (BAU) (for stable recognition of AT base pairs) and N=S (for recognition of TA inversions). We find that the introduction of a duplex mismatch destabilises the C+.GZ, T.AZ and G.TZ triplets. A similar effect is seen with BAU.AZ triplets. In contrast, other base combinations, based on non-standard triplets such as C.AZ, T.TZ, G.CZ and A.CZ are stabilised by the presence of a duplex mismatch. In each case S binds to sites containing duplex mismatches better than the corresponding Watson-Crick base pairs.     

MicroRNA-133a regulates DNA methylation in diabetic cardiomyocytes

We tested the hypothesis that miR-133a regulates DNA methylation by inhibiting Dnmt-1 (maintenance) and Dnmt-3a and -3b (de novo) methyl transferases in diabetic hearts by using Ins2(+/-) Akita (diabetic) and C57BL/6J (WT), mice and HL1 cardiomyocytes. The specific role of miR-133a in DNA methylation in diabetes was assessed by two treatment groups (1) scrambled, miR-133a mimic, anti-miR-133a, and (2) 5mM glucose (CT), 25mM glucose (HG) and HG+miR-133a mimic. The levels of miR-133a, Dnmt-1, -3a and -3b were measured by multiplex RT-PCR, qPCR and Western blotting. The results revealed that miR-133a is inhibited but Dnmt-1 and -3b are induced in Akita suggesting that attenuation of miR-133a induces both maintenance (Dnmt-1) - and de novo - methylation (Dnmt-3b) in diabetes. The up regulation of Dnmt-3a in Akita hearts elicits intricate and antagonizing interaction between Dnmt-3a and -3b. In cardiomyocytes, over expression of miR-133a inhibits but silencing of miR-133a induces Dnmt-1, -3a and -3b elucidating the involvement of miR-133a in regulation of DNA methylation. The HG treatment up regulates only Dnmt-1 and not Dnmt-3a and -3b suggesting that acute hyperglycemia triggers only maintenance methylation. The over expression of miR-133a mitigates glucose mediated induction of Dnmt-1 illustrating the role of miR-133a in regulation of DNA methylation in diabetes.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

Bis-4-aminoquinolines: novel triple-helix DNA intercalators and antagonists of immunostimulatory CpG-oligodeoxynucleotides

Six dimeric 2-(2-naphthyl)quinolin-4-amines with a linker between the amino groups and eight dimeric 2-(4-anilino)quinolin-4-amines linked between the anilino groups were synthesized and evaluated for their interaction with duplex/triplex DNA's and as antagonists of immunostimulatory oligodeoxynucleotides with a CpG-motif (CpG-ODN). The most powerful triple-helix DNA intercalator known to date, with high affinity toward T.A.T triplets and triplex/duplex selectivity, was found. The potent antagonism of immunostimulatory CpG-ODN by several bis-4-aminoquinolines is not related to their DNA interactions.     

Nonenzymatic sequence-specific cleavage of duplex DNA via triple-helix formation by homopyrimidine phosphorothioate oligonucleotides

Phenanthroline was attached covalently to the 5′-terminus of the unmodified and modified (3′-terminal phosphorothioate) oligonucleotide sequences, TTTTTTCTTCTCTTTCC (OP-17 mer) and TTTTTTTCTTCTCTTTCsC (OPRp-17 mer or OPSp-17 mer) via a phosphoramidite bond. Simian virus 40 DNA contains a single target site for these oligonucleotides. In the presence of copper ions, the efficient double-stranded cleavage at 37 °C and pH 7.0 was observed by agarose gel electrophoresis. The asymmetric distribution of the cleavage sites on the two strands revealed that the cleavage reaction took place in the minor groove, even though the linker was located in the major groove. Of particular interest are the 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp), which were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes and antisense nucleic acid based anti-viral therapies, which could be used as tools in cellular biology.

The 3′-terminal phosphorothioate oligonucleotide-phenanthroline derivatives (Rp or Sp) were found to have cleavage activities of the same order as for the oligonucleotide phenanthroline (OP-17 mer). Furthermore, the OPSp-17 mer was intact after incubation in 10% fetal bovine serum for 24 h, whereas, the OPRp-17 mer was slightly more unstable than the OPSp-17 mer. However, the OP-17 mer was completely degraded. An increased resistance to nucleases has been observed by the introduction of phosphorothioate groups on the 3′-terminus of oligonucleotide-phenanthroline derivatives. This stabilization should help us to design much more efficient chemical recognition enzymes, which could be used as tools in cellular biology.     

利用CRISPR-Cas9基因编辑技术敲除H9c2细胞Tudor-SN 基因对细胞周期的阻滞及增殖的抑制

基因特异性敲除的细胞常用于生物学研究。近些年兴起的CRISPR-Cas9 （clustered regularly interspaced short palindromic repeats-Cas9 nuclease）基因编辑技术越来越广泛地用于基因特异性敲除的实验中。本实验通过利用CRISPR-Cas9基因编辑技术,实现对大鼠心肌H9c2细胞Tudor-SN（tudor staphylococcal nuclease）基因的敲除,并观察其对H9c2细胞周期及增殖的影响。选择PX462质粒为载体,利用软件设计能特异性识别H9c2细胞Tudor-SN 基因第2个外显子的上下游sgRNA（single-guided RNA）,构建1对重组质粒。随后,将这对质粒共同转入H9c2 细胞中,再挑选阳性单克隆细胞进行培养。Western 印迹鉴定其敲除效果,并用敲除成功的细胞株通过流式细胞术及CCK-8（cell counting kit 8）实验进行细胞周期和增殖的检测。Western 印迹结果显示,在阳性细胞中,Tudor-SN 蛋白不表达,成功实现了对Tudor-SN基因的敲除。流式结果显示,Tudor-SN基因敲除（KO）细胞发生了G1期阻滞。G1期细胞占比由H9c2 野生型（WT）细胞的54.28%±0.21% 升高到KO细胞的61.96%±0.40%（*P< 0.05）。CCK-8实验结果显示,KO细胞的增殖速率减慢。生长第6 d的A值由WT细胞的2.82±0.03降低到KO细胞1.85±0.19（*P< 0.05）。本实验成功构建了H9c2 细胞Tudor-SN 基因敲除细胞株,并检测到Tudor-SN 基因敲除对细胞周期的阻滞及增殖的抑制,为研究Tudor-SN基因对心肌细胞功能的调控提供了便利的工具及研究的基础。     

DNA triple-helix formation on nucleosome core particles. Effect of length of the oligopurine tract.

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.     

Magnetic ordering of DNA liquid crystals

Sonicated calf thymus DNA with an average length of approximately 100 base pairs has been found to form a cholesteric liquid crystal at a concentration of approximately 250 mg of DNA/mL of solution. Immediately after preparation, small ordered domains of a few micrometers are formed, resulting in an opaque solution. This liquid crystal can readily be oriented in the magnetic field of an NMR magnet, resulting in a clear birefringent phase. The DNA molecules align with their helix axes perpendicular to the field so that the cholesteric pitch axis was parallel with the field. A pitch length of approximately 2.5 microns for the cholesteric phase was determined both from optical measurements (optical light rotation) and from NMR measurements (solvent diffusion). The observation that DNA molecules can be magnetically oriented opens up new possibilities for studying the structure and dynamics of the aligned DNA molecules.     

Designed DNA molecules: principles and applications of molecular nanotechnology

Long admired for its informational role in the cell, DNA is now emerging as an ideal molecule for molecular nanotechnology. Biologists and biochemists have discovered DNA sequences and structures with new functional properties, which are able to prevent the expression of harmful genes or detect macromolecules at low concentrations. Physical and computational scientists can design rigid DNA structures that serve as scaffolds for the organization of matter at the molecular scale, and can build simple DNA-computing devices, diagnostic machines and DNA motors. The integration of biological and engineering advances offers great potential for therapeutic and diagnostic applications, and for nanoscale electronic engineering.     

From liquid crystals to DNA nanoconstructions

This paper reviews the data obtained in our laboratory on the physicochemical properties of liquid-crystalline dispersions formed by double-stranded nucleic acids (DNA and RNA) and the specific features of their behavior in quasinematic layers. The basic data obtained in this field have been used as the background for elaboration of the DNA nanoconstructions carrying various guest molecules (chemical substances or biologically active compounds). Two theoretically feasible approaches to designing DNA nanoconstructions are compared. The unique properties of these nanoconstructions determining the area of their application are described.