Design Of Nucleic Acid Structure And Its Application In MicroRNA Analysis And MRNA Expression Regulation

Nucleic acids,including deoxyribonucleic acid(DNA)and ribonucleic acid(RNA),are a class of essential biopolymers for the organism.With the further study of nucleic acids,the stable DNA double helix structure makes it not only the carrier of genetic information but also an important biological material,which has been used in the construction of a variety of nanoscale assemblies.On the other hand,RNA is usually flexible and single-stranded,with many types which play vital roles in the transmission of genetic information and regulating gene expression in organisms.In this paper,we propose the design of nucleic acid structures,including DNA nanowire and RNA hairpin,which can be applied to the microRNA(miRNA)in vitro analysis and mRNA expression regulation.In recent years,it has been challenging to develop labeling-and amplifying-free methods to achieve the logical computation of low-level miRNA molecules.Therefore,in Chapter 2,we proposed a strategy for the logical operation of miRNA using plasmid DNA-derived double-stranded DNA(dsDNA)nanowire structure.First,let-7d was used as the model of the DNA nanowire strategy,which proved that our nanowire system had good response sensitivity and selectivity.Subsequently,the operation of miRNA logic gates demonstrated the programmability of the constructed plasmid DNA-derived nanowire system.Finally,we designed three pairs of DNA nanowires with different lengths to demonstrate the effectiveness of this strategy for the simultaneous analysis of multiple miRNAs.Compared with traditional miRNA analysis methods,our method requires no additional chemical labeling or amplification,which can obtain readout signals directly from agarose gel.Given the good response of logic gates to miRNA molecules at low concentrations,our DNA nanowire strategy can provide a simple method for the simultaneous analysis of multiple miRNAs.Therefore,the plasmid DNA-derived nanowire strategy may offer a powerful tool for simultaneously detecting of various miRNAs and show potential for large-scale complex DNA nanostructure assembly and clinical diagnosis.On the other hand,target accessibility is an energy-based criterion related to the secondary structure of target RNA,which has been widely used to evaluate the effect of structural RNA on the silencing capacity of RNA-induced silencing complex(RISC).In Chapter 3,we found another non-target accessible path followed by RISC by designing the secondary structure of target RNA.We found that the RISC system can efficiently silence target RNAs fully folded into the hairpin structure with the help of the 3’ tail of the guide RNA(miRNA or short hairpin RNA(shRNA)).This finding is different from the rule that the secondary target structure will block RISC silencing in target accessibility,indicating that the RISC system does not rely on the secondary structure of the target when silencing mRNA.We also found that when interacting with different 3’ tail sequences of guide RNAs.RISC showed different effectiveness in unfolding the secondary target structure.The discovery of a RISC silencing pathway independent of the target secondary structure and the function of the 3’ tail will advance the investigation of the underlying mechanism of RISC silencing mRNA and open up new avenues for target prediction and small interfering RNA(siRNA)/shRNA drug development.In Chapter 4,we design an shRNA structure that can reduce the off-target effect of shRNA by shortening the pairing length of the 3’ tail of its antisense strand with the target sequence without damaging the silencing efficiency of the shRNA.This strategy can effectively improve the gene silencing specificity of shRNA and is more feasible and straightforward than previous studies to reduce the off-target effect.It only needs to shorten the length of pairing between the 3’ region and the target and is not limited by the sequence of the 3’ region of the antisense strand.These studies provide new rules that can significantly improve siRNA/shRNA design.Therefore,our method offers new ideas for the design of shRNA and siRNA,simplifies the sequence constraints in shRNA and siRNA drugs to some extent,and broadens the application and prospect of shRNA and siRNA as therapeutic,diagnostic tools in medicine.In Chapter 5,we achieved precise regulation of gene expression levels in mammals by designing an RNA hairpin element located in the 5’ untranslated region of mRNAs.First,we found that the thermodynamic stability and GC content of the designed hairpin elements affected their gene expression regulation ability.Second,isolation of the start codon in the double-strand structure of the hairpin element can hinder ribosome recognition of the start codon.Third,the hairpin elements and the upstream open reading frame(uORF)sequence have a synergistic effect on mRNA translational inhibition.This strategy demonstrates that the application of defined synthetic RNA structures in the regulation of mRNA translation can modulate protein expression levels in mammals.