The Influence Of Nucleic Acid Secondary Structure On Molecular Diagnostics And The Related Application Research

Single-stranded nucleic acid can form secondary structures by internal complementary sequence.The secondary structures of nucleic acids have their critical biological significance in vivo and in vitro.The tRNA(transfer-RNA)formed trefoil structures which help transport amino acids into the ribosome and synthesize peptide chains depending on mRNA.Secondary structure in mRNA can assist in correct splicing as well.DNA is single-stranded during replication,transcription and repair in vivo.For example,the folded G-quadruple plays a role in the extension of telomeres,and the secondary structure of viral single-stranded DNA is involved in protein recognition.In addition to the important role in vivo,the secondary structures of nucleic acids also have vital bioengineering functions.Self-folding molecular beacons are used to detect nucleic acids,and aptamers that fold into more complex structures are leveraged to identify proteins;To note,the secondary structures of templates impede PCR,Sanger sequencing and hybridization reactions as well.With aging of the population and the improvement of awareness of medical care,the demands for precision medicine are increasing.Cancer has become the number one enemy endangering human life and health.Without the analysis of the genome,transcriptome and proteome,it is impossible to understand the occurrence,development,prevention and treatment of the disease.Nucleic acid detection methods enable personal genome analysis(SNP,rare mutation and methylation profile etc.),provide personalized diagnosis and far exceeds other diagnostic methods in accuracy and information density.The methods are roughly classified into groups of hybridization,PCR,sequencing technology and mass spectrometry.PCR,sequencing and hybridization technology which were focused on in our research,are closely related to the thermodynamics of nucleic acid.However,the secondary structures have an adverse effect on hybridization,PCR and Sanger sequencing.To obtain deep and correct understanding of the influence imposed by structures would benefit biological research and clinical testing.Nearest-neighbors thermodynamics(NNT)proposed that the stability of base pairs depends on the nearest bases.Based on this theory,programs and algorithms for predicting structure and hybridization have been developed.The structure prediction and thermodynamic calculations is of great significance to develop the methods of eliminating the adverse effects of structures and guide the experimental design.First,we developed a new method that can eliminate the secondary structures in templates to rescue the hybridization.Both external and internal oligonucleotides(disruptors)work well in eliminating the original secondary structures.The methods were efficient,economical and general.Structure prediction based on the nucleic acid thermodynamics guided the design of disruptors,which greatly increased the efficiency and success rates of experiments.Next,we confirmed that the secondary structures inhibit PCR,and hihger the stability of the secondary structures are,the stronger the inhibitory effects become.Further,we explored and proposed a new mechanism for this inhibition:the endonuclease of Taq polymerase digests the structured template,thereby generating a truncated PCR product and reducing the PCR efficiency.Furthermore,in order to solve the inhibition imposed by structure,we developed a new,general and efficient disruptor-based strategy(oligonucleotides)in addition to the methods such as increasing the annealing/extension temperature and reducing the Mg2+concentration.A pair of oligonucleotides complementary to templates eliminates the secondary structure by strand-displacement and restores the normal PCR.The integrity of rAAV ITR is essential for virus replication and packaging.Therefore,analysis of its sequence content is highly necessary for gene therapy.However,amplification and Sanger sequencing are inhibited by the highly stable T-shaped structure formed in ITR,and the disruptors were used to solve the inhibition with success.Additionally,artifactual sequencing caused by the secondary structures of the templates was confirmed for the first time in Sanger sequencing.Palindromic sequences and erroneous mutations took place of the supposed sequence at 3’ end.In our research,it was proposed that the artifacts were resulted from the endonuclease activity of Taq polymerase.We believed that the research focused on the influence of secondary structure on PCR,Sanger sequencing and hybridization,and the methods developed to alleviate the adverse influence will enable molecular diagnostics to serve better in biological research and clinical testing.