Self-assembly of porphyrins on nucleic acid templates

The cis and trans isomers of dicationic bis(4-N-methylpyridyl)diphenylporphine show a much greater tendency to aggregate than similar tetracationic porphyrins. Upon binding to nucleic acids these aggregating dicationic porphyrins form long-range structures on the polymer template giving intense circular dichroism signals whose profile reports the helical sense of the DNA.     

Dynamic assembly of primers on nucleic acid templates

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of ~1 μM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.     

Kinetics of synthesesis of polymeric dAT on oligomeric templates

A model is developed to explain the following experimental observations: oligomers of the alternating DNA copolymer dAT serve as templates for the enzymatic synthesis of macromolecular dAT; the rate of polymer synthesis with a template oligomer of given length increases to a maximum and then decreases as the temperature increases; the temperature for the maximum rate of synthesis increases with the length of the template. These observations were interpreted in terms of the repeated “slipping” of the oligomer along the product strand to expose template sites. This communication extends this idea. We suppose that the rate-limiting step in the polymerization is either the attachment of the substrate to the exposed template site or the synthesis of the phosphodiester bond. Then the rate of synthesis is proportional to the equilibrium concentration either of exposed template sites or of template sites occupied by substrate molecules. These concentrations are evaluated in terms of a simple theory of the helix–coil transition. The model qualitatively reproduces the dependence of the rate of synthesis on template length and temperature and predicts rates of synthesis in approximate agreement with experiment.     

An autonomous fueled machine that replicates catalytic nucleic acid templates for the amplified optical analysis of DNA

Here we describe a protocol for the amplified detection of a target DNA using a DNA/FokI-based replicating cutting machine. The protocol is based on the design of a sensing hairpin oligonucleotide that is opened upon hybridization with the analyte DNA. The endonuclease FokI binds to the double-stranded complex and cleaves it to a "cutter" unit. The "cutter" unit reacts with a fuel oligonucleotide to generate and amplify the signal. The fuel molecule is an oligonucleotide in a hairpin configuration with a fluorophore/quencher pair attached to the 5' and 3' ends. Formation of the duplex between the cutter and the fuel leads to the scission of the duplex by FokI, leading to a second, replicated "cutter", a fluorescent waste product, and to the regeneration of the original "cutter" unit. The autonomous replication of the "cutter" unit, as a result of the primary recognition of the analyte DNA, leads to the amplified fluorescent detection of the analyte DNA with a sensitivity limit of 1 x 10(-14) M. The operation of the machine and the sensing process are monitored by the fluorescence generated by the waste product. Here we apply the protocol, which takes about 2 h to complete, to analyze a Tay-Sachs genetic disorder mutant DNA.     

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.     

Allosteric nucleic acid catalysts

Endowing nucleic acid catalysts with allosteric properties provides new prospects for RNA and DNA as controllable therapeutic agents or as sensors of their cognate effector compounds. The ability to engineer RNA catalysts that are allosterically regulated by effector binding has been propelled by the union of modular rational design principles and powerful combinatorial strategies.     

Fluorinated peptide nucleic acid

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.