Novel Hoogsteen-like bases for configurational recognition of the T-A base pair by DNA triplex formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides binding duplex A-T or G-C base-pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺·G-C triplets. Here we report the successful modeling of novel unnatural nucleosides that recognize the T-A DNA base pair by Hoogsteen interaction. Since the DNA triplex can be considered to assume an A-type or B-type conformation, these novel Hoogsteen nucleotides are tested within model A-type and B-type conformation triplex structures. A triplet consisting of the T-A base pair and one of the novel Hoogsteen nucleotides replaces the central T·A-T triplet in the triplex using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration. The entire triplex is energy minimized and the presence of any structural or energetic perturbations due to the central triplet is assessed with respect to the unmodified energy-minimized (T·A-T)₁₁ proposed starting structures. Incorporation of these novel triplets into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets. The plan is to produce a series of Hoogsteen-like bases that preferentially bind the T-A major groove in either an A-type or B-type conformation. Selective recognition of the T-A major groove with respect to the G-C major groove, which presents similar keto and amine placement, is also assessed with configurational preference. Evaluation of the triplex solution structure by using these unnatural bases as binding conformational probes is a prerequisite to the further design of triplet forming bases. © 1996 John Wiley & Sons, Inc.     

Novel Hoogsteen-like Bases for Recognition of the C-G Base Pair by DNA Triplex Formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides that bind duplex A-T or G-C base pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺ ·G-C triplets. Here we report the successful modelling of novel unnatural nucleosides that recognize the C-G DNA base pair by Hoogsteen-like major groove interaction. These novel Hoogsteen nucleotides are examined within model A-type and B-type conformation triplex structures since the DNA triplex can be considered to incorporate A-type and/or B-type configurational properties. Using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration, a triplet comprised of a C-G base pair and the novel Hoogsteen nucleotide, Y2, replaces the central T·A-T triplet in the triplex. The presence of any structural or energetic perturbations due to the central triplet in the energy-minimized triplex is assessed with respect to the unmodified energy minimized (T·A-T)₁₁ starting structures. Incorporation of this novel triplet into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.     

A probabilistic approach to microRNA-target binding

Elucidation of microRNA activity is a crucial step in understanding gene regulation. One key problem in this effort is how to model the pairwise interactions of microRNAs with their targets. As this interaction is strongly mediated by their sequences, it is desired to set-up a probabilistic model to explain the binding preferences between a microRNA sequence and the sequence of a putative target. To this end, we introduce a new model of microRNA-target binding, which transforms an aligned duplex to a new sequence and defines the likelihood of this sequence using a Variable Length Markov Chain. It offers a complementary representation of microRNA–mRNA pairs for microRNA target prediction tools or other probabilistic frameworks of integrative gene regulation analysis. The performance of present model is evaluated by its ability to predict microRNA–target mRNA interaction given a mature microRNA sequence and a putative mRNA binding site. In regard to classification accuracy, it outperforms two recent methods based on thermodynamic stability and sequence complementarity. The experiments can also unveil the effects of base pairing types and non-seed region in duplex formation.     

Regulation of p27Kip1 by miRNA 221/222 in Glioblastoma

Levels of p27Kip1, a key negative regulator of the cell cycle, are often decreased in cancer. In most cancers, levels of p27Kip1 mRNA are unchanged and increased proteolysis of the p27Kip1 protein is thought to be the primary mechanism for its down-regulation. Here we show that p27Kip1 protein levels are also down-regulated by microRNAs in cancer cells. We used RNA interference to reduce Dicer levels in human glioblastoma cell lines and found that this caused an increase in p27Kip1 levels and a decrease in cell proliferation. When the coding sequence for the 3'UTR of the p27Kip1 mRNA was inserted downstream of a luciferase reporter gene, Dicer depletion also enhanced expression of the reporter gene product. The microRNA target site software TargetScan predicts that the 3'UTR of p27Kip1 mRNA contains multiple sites for microRNAs. These include two sites for microRNA 221 and 222, which have been shown to be upregulated in glioblastoma relative to adjacent normal brain tissue. The genes for microRNA 221 and microRNA 222 occupy adjacent sites on the X chromosome; their expression appears to be coregulated and they also appear to have the same target specificity. Antagonism of either microRNA 221 or 222 in glioblastoma cells also caused an increase in p27Kip1 levels and enhanced expression of the luciferase reporter gene fused to the p27Kip1 3'UTR. These data show that p27Kip1 is a direct target for microRNAs 221 and 222, and suggest a role for these microRNAs in promoting the aggressive growth of human glioblastoma.     

Solution structure of a dsDNA:LNA triplex

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5′-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19°C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.