| There are two main ways in which DNA exists in the human body:one is the well-known DNA double helix structure,which is the B-DNA structure.They are present in the nucleus,carrying the first genetic code,and precise base pairing.The law transmits the genetic information to the progeny DNA accurately,which is the main genetic material in the human body;the other is the promoter region located outside the nucleus,telomere,or some nuclear-expressing genes,and certain proteins in the cytoplasm Combined,these DNA molecules usually do not exist in the right-handed double helix structure.We call it non-B-DNA.Up to now,we have found more than10 non-B-type DNA species.This paper is mainly for DNA i-Motif,DNA G-quadruplex and DNA hairpin-type structures were subjected to surface enhanced Raman spectroscopy(SERS)analysis.Although as early as 1953,Watson and Crick discovered the right-handed double helix structure of DNA,the research topic of detecting nucleic acid structure has always been a hot spot of concern.In the detection of double-stranded B-DNA,we often rely on PCR amplification combined with second-generation shotgun sequencing technology to analyze the base sequence and content of double-stranded DNA.It is different for non-B-DNA,because its genetic information is different from B-type DNA from base order and content,but it shows important biological functions depending on its structure.Therefore,the detection of non-B-DNA is mainly for its topological folding structure.X-ray crystal diffraction(XRD)and nuclear magnetic resonance(NMR)technology have become the main detection methods,but DNA molecules are different from other organic small molecules.The natural topological folding structure is controlled by conditions such as concentration,temperature,and solution environment as well as salt ions.Therefore,the high purity,high concentration and crystallization process required by these two techniques make the detection method time-consuming and labor-intensive.And the detection cost is extremely expensive.And some other simple test methods,CD spectrum,UV,etc.can only provide simple structural information,and can not be used as the main means to study the details of DNA molecular structure.Compared with the above methods,SERS has many advantages such as simplicity,sensitivity,short time consumption,trace quantity,etc.,and the fingerprint information inherited from Raman spectroscopy can correspond to the detailed information of nucleic acid structure.Therefore,it can be used as an effective means for detecting nucleic acids.However,there are many difficulties in the application of SERS to nucleic acid detection.This paper has designed a new method for these difficulties to realize the analysis of DNA spatial structure,including the following four parts:a.We have established a simple and reliable method to analyze the four-molecular DNA i-motif structure by SERS rapid detection.We attempted to use aluminum ions with three positive charges to induce aggregation of silver nanoparticles to form high quality"hot spots",resulting in highly reproducible SERS spectra.We successfully detected the structural features of DNA i-motif.For the first time,the formation of DNA i-motif structures was detected and analyzed using SERS spectroscopy.In addition,the number of base pairs of DNA i-motif structures is quantified based on the relative intensity of the SERS peaks in order to estimate the relative stability of the structure.This approach significantly improves the range of SERS applications,not only for single-stranded DNA and single-base detection,but also for detecting multi-molecular complex DNA molecules.b.Rapid acquisition of structural information for DNA G-quadruplexes using our new approach.Our method is highly sensitive and reproducible.Indeed,for the first time,we have reported high-intensity peaks corresponding to dG and dA ring breathing variations located in the G-quartet plane.In addition,we also obtained a series of neat and enhanced bands of G-quadruplexes representing particular structural features,such as hydrogen bonds,glycosidic band angles,and layers of G-quartets,which could be used to distinguish structural polymorphisms in detail.Our results strongly suggested that the G-quadruplex adopted an appropriate position within the Ag IANPs,resulting in the enhancement of the intensity of those bands that correspond to the base ring breathing and hydrogen bonds in the G-quartet.Therefore,the stability trend of G-quadruplex and the number of layers in the G-quadruplex can be quantitatively evaluated by measuring the relative intensity of SERS bands assigned to Hoogsteen hydrogen bonds.The present study provides a new way for label-free characterization of G-quadruplexes and further work on the different DNA conformations and the interactions between DNAs and their ligands in the organism.c.we have successfully developed the method applicable for reliable,sensitive and truly label-free determination of double-strand DNA hybridization with carefully designed DNA strands in aqueous solutions.Our method not only has been proved efficacious in the precise determination of G-C content in hairpin dsDNA but also reveals the stability rules with regard to the base stacking by normalizing the SERS peak intensity at 959 cm~-11 representing the deoxyribose of nucleotides.With the ability of highly sensitive and reliable determination of double-strand DNA,we are able to apply this method to get a clear observation of the base mismatches in a double helix DNA and identification about the position of the wrong-paired base.d.With the introduction of dichloromethane as an interfacial agent in the aqueous solution,we modified the existed SERS method to make it a more novel method for detection of long-chain double strand DNA.Under the newly-developed method,for the first time,indiscriminate quantitative measurements of base content in DNA strand of different length were accessible due to the enlarged space between the metal nanoparticles.Also,further experiments focusing on the detection of single-base mutation in the gene BIGH3 proved the applicability of this method to get rapid and precise information in the long-chain double strand DNA.With such improvement,this method would be of great value in gene selection and clinical research. |
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