Small Molecules And The Role Of The Dna Bulge Structure And Efficient Dna Enzyme Screening

DNA is a kind of important biological large molecules that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information, also as templates for replication and transcription. Design and synthesis of compounds with activity of genetic regulation, especially capable of selective binding to noncanonical base pairs, is one of the most important fields in biochemistry and will provide precursory research basis for the development of genetic regulation drugs.Bulged structure in nucleic acid is one of the important motifs in DNA recognition. Its extra unpaired nucleobases are capable of forming complexes with nucleic acid-bindingproteins as well as acting as the binding site for small molecules. They are believed to be the intermediates in the fragment shift mutagenesis inducing some genetic diseases. Bulge structure in nucleic acid has been shown to play a significant biological role in protein binding recognition, frameshift mutation, imperfect homologous recombination by repair enzymes, naturally occurring antisense RNA and expansion of triplet repeats during DNA synthesis.We studied the binding affinity and binding selectivity between R/S binaphthalene aminosugar compounds with different DNA sequences using fluorescence spectrum, and found that they had selective binding affinity with bulged structure. We also studied the binding affinity and binding selectivity between phenothiazine aminosugar compound with different DNA sequences using UV and CD spectrum, and SPR, which showing the best binding affinity with three-nucleotides bulged structures. Using SPR, we got the dissocation constants of R/S binaphthalene aminosugar compounds and phenothiazine aminosugar compound with three-nucleotides bulged structure.Small molecules with bulged structure binding affinity can stimulation triplet repeat expansion in vitro. Using the triplet repeat expansion system, we found that R/S binaphthalene aminosugar compounds, phenothiazine aminosugar compound and cis/trans azabenzene aminosugar compounds showed different levels of stimation on triplet repeat expansion. Trans-azabenzene aminosugar compound can stimulate the expansion, while after UV irradiation cis-azabenzene aminosugar compound restrained the expansion. Using phenothiazine aminosugar compound and DNA polymerase I, we studied the mono-, double and other triplet repeat expansion in vitro. All the sequeces extended during the replication, and extensions were stimulated by the compound. The sequences with relatively unstable secondary structures slipped in a way more than that of the sequences which could form stable secondary structures.Using DDI compound, we checked the influence of different length of template, non-repeat sequence and reaction temperature on expansion of triplet repeat sequence. In a short reaction time, template with less repeat numbers extended longer. Non-repeat sequence interrupted the continuity of triplet repeat sequence and influenced on the expansion in vitro. Increasing reaction temperature was propitious to slippage.In the same reaction conditions, we studied phenothiazine aminosugar compound, R/S binaphthalene aminosugar compounds, DDI, ent-DDI and an analogue of neocarzinostatin chromophore on stimulation effect of triplet repeat DNA synthesis in vitro and found that they had different effect. Comparing binding ability of bugled structure and stimulation effect of different small molecules will be benefit to design and synthesis of small molecules which can target to bugled structure and be helpful to study the triplet repeat expansion mechanism.Deoxyribozyme also known as DNAzymes or DNA enzymes, refer to single-stranded DNA molecules with catalytic capabilities. DNAzymes are generated de novo by in vitro selection—a powerful and yet simple technique that has been routinely used to isolate extremely rare DNA or RNA sequences with a function of interest (e.g. ligand-binding or catalysis) from an extraordinarily large population of single-stranded DNA or RNA molecules.The catalytic scope of nucleic acid chemistry can be well illustrated by a wide range of chemical reactions that are catalyzed by existing NAEs, for examples, RNA-cleavage, DNA ligation and porphyrin metallation. Its catalytic competency, however, is in most cases several orders of magnitudes less efficient than the protein counterpart. Considering that a typical in vitro selection experiment only requires the nucleic acid species in a large array of random sequences to process one single substrate in one round of selection, we propose that if a library is forced to mediate the catalysis of more than one reactions in a short period of time, only the highly efficient NAEs with high multiple turnovers would survive and be isolated. We chose RNA-cleavage DNA as the model reaction and catalytic platform and a three-way RNA as subtrate in our selection experiment.The extension was carried out with 200 pmol DNA pool containing a N60 random sequence, which was ligated to the three-way RNA substrate with a DNA junction. The oligos with catalytic activity was amplicated by PCR and into another round of selection. After 12 rounds, we have got several DNAzymes with RNA-cleavage catalytic activity.There are bulged structure in the formation of DNAzyme active center. Small molecules which are able to bind to DNA bulged structure can also be used to bind to DNAzyme to stabilize the formation and regulate its activity.