Development And Applications Of L-nucleotide-based Novel Functional Nucleic Acid Fluorescent Probes

With deeper understanding and study of nucleic acids,an increasing amount of simple and low-cost nucleic acid-based fluorescence probes have been developed and play an important role in biochemical analysis.Functional nucleic acids(FNAs),one of the important productions from nucleic acid molecular technique,their emergence break the limitation that the early stage fluorescence probes were mainly constructed for the function on a nucleic acid molecule level,such as cancer gene diagnosis and the analysis of gene expression.FNAs mainly refers to the nucleic acids possessing recognition or catalytic functions.They can be obtained from an in vitro screening technology,and immensely broaden the analytes’ scope of nucleic acid-based fluorescence probes.Due to easy chemical synthesis and flexible design compared with antibodies and proteases,nucleic acid-based fluorescence probes display good reproducibility,high sensitivity and selectivity.However,the performance of nucleic acid-based fluorescence probes is limited in complex biological and environment samples for several reasons.First,the stability of nucleic acids are compromised due to the enzyme susceptibility.Second,in complex environment,nonspecific protein absorption and non-target DNA or RNA hybridization lead to off-target effect and false positive/negative signals.In order to solve the above-mentioned problems,nanomaterials and self-assembly strategies are involved in the development of nucleic acid-based fluorescence probes.However,the cytotoxicity of nanomaterials needs to be considered,and the effect of nanomaterials on the function of nucleic acids needs to be evaluated.Therefore,the stability and antijamming capability of nucleic acid-based fluorescence probes are an important issue to be addressed urgently.Based on the concept of enantiomer,unnatural peptides and proteins constructed by D-amino have been developed.In nature,L-protease can be active with corresponding L-substrate,and possess catalytic capability.The reports have proved that D-protease can recognize its D-substrate with catalytic activity the same as D-protease dose.Also,D-prostease can’t function with L-substrate,vice versa.This property can be called as reciprocal chiral substrate specificity.As similar as proteins,natural nucleic acids are all constructed by D-nucleotides.Based on the concept of enantiomer,L-nucleotides have been synthesized for a novel unnatural nucleic acids,(L-nucleic acids).Compared with D-nucleic acids,L-nucleic acids possess similar physical and chemical properties.L-nucleic acids also follow the principle of Watson-Crick base pairing,but they can’t hybridize with D-nucleic acids.Meanwhile,benefiting from the stereoselectivity in living bodies,L-nucleic acids are insusceptible to nuclease degradation and nonabsorption with proteins.Therefore,L-nucleic acids show excellent biostability and antijamming capability.Based on the reciprocal chiral substrate specificity,we design and synthesize L-nucleic acids which possess special function by L-nucleotide.Combining the the properties of FNAs and L-nucleic acids,functional L-nucleic acids can recognize various targets with high affinity and specificity,and are insensitive to nuclease digestion,protein adsorption and D-DNA hybridization.Therefore,we construct a series of simple and bio-stable fluorescence probes with excellent antijamming capability for the detection of small molecules and metal ions crucial in living systems,and successfully applying them in complex samples,such as river water,serum samples and living cells.Details are as follows:(1)In Chapter 2,based on existing aptamer of achiral small molecules,we synthesize the mirror form ones(L-aptamers)and prove that they can also specifically bind to their targets and displays excellent biostability.Malachite green(MG),toxic to human beings and animals,is used to be an antibacterial agent in aquaculture,causing the food safety problems associated with fishes.For this reason,the RNA aptamers of MG are chosen as a model to construct L-aptamer-based "turn-on" fluorescence probes for MG detection.This MG probe can response well to MG with a linear response in the range of 0.1～1 μM and a detection limit of 65 nM in aqueous solution.Meanwhile,this MG probe can display a good performance in fish tissue extractions and lake water samples as well.Moreover,we also prove the feasibility of the probe for ethylenediamine(EA)and show the universality of this L-aptamer-based strategy for constructing fluorescence probes.(2)In Chapter 3,taking advantage of the specific binding between T-rich DNA sequences and mercury ions or C-rich DNA sequences and silver ions,L-aptamer-based fluorescence probes are constructed for the detection of mercury ions or silver ions.We prove that T-rich DNA or C-rich DNA can recognize mercury ions or silver ions to form hairpins structures("T-Hg2+-T" form or "C-Ag+-C" form),and are all insusceptible to nuclease degradation.Fluorophore and quencher are labeled at the both ends of T-rich DNA sequences or C-rich DNA sequences.In the presence of mercury ions or silver ions,these probes will bind to corresponding metal ions and form hairpins,leading to fluorescence quenching.This Hg2+ probe can response well to Hg2+with a linear response in the range of 25～250 nM and a detection limit of 20 nM in aqueous solution,and the Ag+ probe shows a linear response in the range of 10～100 nM and a detection limit of 6.6 nM.Both of the two probes are sensitive,selective and biostable,and the performance in real water samples are as good as in buffers.(3)In Chapter 4,we study on potassium ion and its aptamer.Potassium ions play an important role in living system,due to their biological functions for the regulation of life activities.Therefore,the analysis of potassium ions in physiological conditions is of great importance and necessity.Natural thrombin aptamer(D-TBA),which can fold into pure G-quadruplex forms,possesses nonspecific binding to potassium ions and cytotoxicity with its G-quadruplex form and degradation products.In this paper,we synthesize the D-TBA’s enantiomer(L-TBA)and construct an L-G-quadruplex-based fluorescence probe for potassium ion detection and imaging in living cells.In our experiments,the structure of L-TBA is charactered by circular dichroism(CD),and the performance in aqueous solution and living cells is optimized through different experiment conditions.Due to the anti-protease degradation ability,and nonabsorption with thrombin,other proteins and cells,L-TBA can not only efficiently avoid false positive signals,but also shows almost noncytotoxicity.This L-TBA-based fluorescence probe overcome the drawbacks of D-TBA and displays better imaging performance of potassium ions in living cells.(4)In Chapter 5,according to lead ions-dependent GR-5 DNAzyme,we synthesize the enantiomer(L-GR-5 DNAzyme),which is the mirror form of normal DNAzyme(D-DNAzyme).Utilizing this L-DNAzyme,we construct a "turn-on"fluorescence probe for lead ions detection in complex samples.Based on reciprocal chiral substrate specificity,lead ions-dependent L-DNAzyme can specifically bind with lead ions and cleave its substrate.L-GR-5 DNAzyme are against nuclease degradation,nonspecific absorption with proteins and don’t hybridize with D-DNA.Especially,L-substrate with an L-rA also can maintain its properties for a long perid of time,far better than D-subtrate.With two-quencher strategy,this probe displays excellent biostability,high sensitivity and selectivity with a detection limit of 3 nM,and better function than D-DNAzyme-based probes in 5%fetal bovine serum(FBS)samples(without acidolysis experiment).