The Assembly Of Non-natural Nucleotides And The Structural Properties Of Their DNA Strands

As one of the most basic substances of life,nucleic acid is the material basis of genetic variation in organisms.Nucleotides are the basic building blocks of nucleic acids,involving in almost every important biological function in vivo.It consists of three parts:ribose or deoxyribose,phosphoric acid and nitrogenous bases.Among them,nucleobases are the most important part of nucleotide and play a leading role in the transmission of genetic information.There are five main nitrogenous bases in the organism:adenine?A?,guanine?G?,cytosine?C?,thymine?T?and uracil?U?.All living organisms store and transmit genetic information use the above five bases from generation to generation.However,the genetic"alphabet"is not immutable.In recent years,research on unnatural nucleobases has attracted much attention.The appearance of unnatural bases would increase the types of nucleotides,expand the genetic information system alphabet and improve the diversity of genetic code.The prebiotic assembly of non-natural nucleotides and the structural properties of DNA duplex have important scientific significance in exploring the chemical origin of life from the microscopic level,revealing the chemical mechanism of genetic information transmission and understanding the dynamic structure and function of DNA/RNA in living organisms.In this dissertation,a detailed theoretical study of the prebiotic assembly of nucleotides and the structural properties of DNA duplex containing unnatural nucleobase was performed using molecular dynamics simulations and quantum chemistry methods.The main research results are highlighted as follows:?1?The detailed formation mechanisms of C-ribonucleoside and N-ribonucleoside via the reaction of 2,4,6-triaminopyrimidine?TAP?with?D?-ribose in aqueous solution were explored using density functional theory?DFT?.The calculations indicate that five isomers??,?-furanose,?,?-pyranose and open-chain aldehyde?of?D?-ribose can exist in equilibrium in aqueous solution.In contrast to the cyclic isomers,open-chain aldehyde is most feasible to react with TAP.In general,the formation pathways of C-nucleoside and N-nucleoside proceed in three steps including nucleophilic addition,dehydration and cyclization.The calculated apparent activation energies are 28.8 kcal mol^-1 and 29.2 kcal mol^-1,respectively.It suggests that both C-and N-nucleoside can be formed in aqueous solution,which is in good agreement with the experimental result.By designing explicit water molecule models of participating in the reaction,it was found that the water molecule plays an important"H-bridge"role by the hydrogen atom relay.Finally,a model structure of nucleobase,which will be beneficial for the C-C glycosidic bond formation,is proposed.This study will provide theoretical guidance for the selection of reasonable nucleobase precursors and exploration of the origin of life under prebiotic conditions.?2?Addition of artificial base pair P:Z to the four naturally occurring DNA bases expands the genetic alphabet,yielding an artificially expanded genetic information system?AEGIS?.Here,the structural features of oligonucleotide containing a novel unnatural P:Z base pair and the interaction mechanism of P:Z pair with natural bases were characterized by using both molecular dynamics and quantum chemical calculations.The results show that the incorporation of a novel artificial base pair?P:Z?preserves global conformational feature of duplex DNA except for slightly changes of the local structures.Z-nitro group imparts new properties to the groove width,which widens the major groove.This can be used as a recognition site for proteases.The unnatural oligonucleotides containing mismatched base pairs exhibit low stability.It offers insurance to an efficient and high-fidelity replication.In general,the incorporation of P:Z pair strengthens the stability of the corresponding DNA duplex.The calculated results also show that the thermostability originates from both hydrogen interaction and stacking interaction.Z-nitro group plays an important role in enhancing the stability of H-bonds and stacking strength of P:Z pair.Overall,the present results provide theoretical insights in exploring the artificially expanded genetic information systems and designing novel multifunctional proteins.?3?Fluorescent base analogues are of great importance as sensitive probes for studying the dynamic structures and functions of DNA and RNA in biology.Herein,we explored the structural and photophysical properties of DNA containing4AP/4AP?/4AP⁰:DAP pair in detail,employing both molecular dynamics simulations and quantum mechanics methods.The results show that 4AP:DAP pair is well adapted to the overall B-DNA structure with higher stability and?-stacking abilities.Only two hydrogen bonds that facing to the major groove are formed.But it?s enough to provide sufficient selectivity for the DNA polymerases in the primer extension.The existence of dimethylamino group of 4AP?disturbs the base pairing interaction.The structural overlap of 4AP?and 4AP⁰ with the neighboring adenine only lies in5?-direction which may contribute to the structure distortion from native B-DNA.The photophysical properties of the fluorescent nucleobases and B-DNA duplex were detailed explored as well.It suggests that hydrogen bond interaction between the base and deoxyribose is responsible for the larger Stokes shift of d4AP than 4AP monomer.The stereotactic resistance of dimethyl group in 4AP?causes them deviated from the aromatic rings plane and its fluorescence efficiency greatly reduced even in nucleoside.Additionally,we focused on the effects of the DNA local environment on the fluorescence properties of nucleobases.The base pairing interaction doesn't make much difference for the fluorescence band apart from the slightly red-shifts of spectra maxima.Importantly,identity of the neighboring bases stacked with 4AP has an important effect on the fluorescence band.And the stacking of 4AP with monocyclic pyrimidine bases can better maintain its optical characteristics.Overall,4AP:DAP exhibits a better stability both in structural and photophiscal proterties upon incorporated into DNA environment.We hope our theoretical predictions are helpful for designing new fluorescent probes to study the dynamic structures and functions of DNA/RNA in biology.