Study Of Biomolecular Interactions,Protein And Nucleic Acid Structures Based On Surface-enhanced Raman Spectroscopy

There are multiple interactions between biomolecules,including hydrogen bonds,electrostatic interactions,hydrophilic and hydrophobic interactions,and Van der Waals forces.These interactions are the basis for proteins and nucleic acids to form secondary and even higher structures.Biomolecules such as nucleic acids and proteins need to form various high-level structures in the organisms and interact with other molecules to play their biological functions.Thus,accurate measurement of these interactions is both significant and challenging.Proteins,as important biomolecules,are considered as one of the important components of living systems,and they have rich structural diversity.Amino acids are the basic structural units of proteins.After dehydration and condensation,the carboxyl group of one amino acid and the amino group of one amino acid will form peptide bonds.And a series of amino acids are connected by covalent peptide bonds to form the primary structure of the proteins.The secondary structure of proteins is formed when the polypeptide chains fold and coil regularly in space.The most common secondary structures include?-helice and?-sheet.When the polypeptide chains are further coiled and folded on the basis of the secondary structure,the tertiary structure of the protein can be formed by the amino acid side chain and disulfide bonds.Protein complex formed by polypeptide chains are known as quaternary structure of protein,such as myoglobin.Nucleic acids as guardians of life's memory,and the carrier of genetic information,usually exist in the form of a pair of closely bound double-stranded molecules,also known as B-DNA.However,50%of genomic DNA in the human body is composed of repeated sequences.They fold into secondary conformations different from the classical B-DNA structures through special intermolecular interactions and intramolecular interactions,including H-DNA,Z-DNA,hairpin DNA,cruciform DNA,G-quadruplex,i-motif and GCGC-quadruplex.In order to deeply understand the structural details of the protein and DNA,continued efforts have been made to develop methods,including mass spectrometry(MS),fluorescence spectrum,X-ray diffraction(XRD),and nuclear magnetic resonance(NMR).However,to date,the above methods still face great challenges in sensitivity,speed,and sample loss,especially in monitoring the conformation of protein and DNA and their dynamic changes in complex samples.The development of surface-enhanced Raman spectroscopy(SERS)opens up a new direction for the detection of protein and DNA,which fully inherits the features of traditional Raman spectroscopy.Due to SERS can provide molecular vibration,rotation,and atomic interaction information in aqueous solution,SERS can be used to study interactions between molecules close to physiological conditions,which greatly improves the detection sensitivity of traditional Raman techniques.Moreover,SERS has been successfully applied in vivo detection.SERS can be performed by two methods:indirect detection and direct detection.Indirect detection is usually used to detect the signal of reporter molecules that are covalently connected to the target molecule.The reporter molecules are generally dye molecules with large Raman scattering cross-sections and can produce enhanced Raman signal.Label-free detection can directly provide fingerprint of molecules themselves.But it is usually limited by the Raman cross section of the molecules,and then affect detection sensitivity.To solve this problem,our group has developed a new substrate(Ag IANPs)containing iodide and aluminum ions,which has extremely high detection sensitivity and has been successfully used in the study of different nucleic acid secondary structures such as DNA hairpin,G-quadruplex,and i-motif.On this basis,this paper use Ag IANPs as substrates,and carry out the following three parts to achieve the analysis of the spatial structure and interaction of biomolecules.?.So far,SERS still has many detection difficulties,including the charge of proteins,the kind of residues,the protein conformation,the microenvironment of substrate,and the interaction between proteins and substrates.Due to some proteins have no chromophore,SERS signals only generated by amino acid residues and amide skeleton,so it is very difficult to obtain strong protein signals.In this part,we use Ag IANPs as substrate to analysis acidic proteins(bovine serum albumin,catalase,?-casein,?-casein,insulin)and basic proteins(myoglobin,lysozyme).Hence,the signal for all proteins were significantly improved compared to Ag IMNPs,Ag NPs+Na₂SO₄,and Ag IMNPs+Mg²⁺as substrates.At least three orders of magnitude enhancement in the detection of acidic proteins was achieved by using Ag IANP than any of Ag NPs reported in literature.And the limit of detection(LOD)of bovine serum albumin is 0.03ng/m L.In addition,the bands corresponding to the residues(disulfide bonds,?-helix,Phe,Trp,Tyr and CH₂)of several proteins were closer to the surface of Ag IANPs and significantly enhanced.This will help us to further monitor the folding and denaturation processes of native proteins.In conclusion,by further improving the silver nanoparticles,we have established a new platform(Ag IANPs)for high sensitivity and label-free detection of native proteins.Thus,Ag IANPs as substrates open a novel way for surface-enhanced Raman spectroscopy(SERS)detection of proteins.?.Based on the excellent performance of Ag IANPs,we further explored the effect of DNA intramolecular interactions on DNA SERS detection.Currently,the SERS spectra generated by various DNA conformations have been well studied.Since the formation of DNA secondary structure is regulated by intramolecular interactions,we selected DNA molecules with different intramolecular interactions as models,including three GCGC-quadruplexes(GC,G₃C,and GCA),a G-quadruplexes(Oxy1.5)and a duplex(ds GC).Among them,GCGC-quadruplexes contains both Hoogsteen hydrogen bonds in G-quadruplexes and Watson-Crick hydrogen bonds in DNA duplex.Our aim is to study the spectral changes of these special interactions in different environments.At the same time,studies have shown that the band intensity,shift are closely related to the interactions between the target molecule and the substrate used.The coexisting chemicals in detection system,impurity and the formation of“hot spot”are considered as important factors affecting the reliability of SERS detection.However,intramolecular interactions,as key factors for the construction of the secondary conformation of DNA molecules,have not been paid enough attention to their influence on Raman spectroscopy.To answer these questions,by comparing the DNA molecules,we obtained the effect of various hydrogen bonds and base stacking presented in different DNA conformations on SERS band intensity,shift and appearance.The information obtained are expected to provide new insights into the SERS detection reliability issue and SERS capability for structural analysis.?.As non-classic DNA structure,G-quadruplexes(G4s)have been shown to exist in chromosomal telomeres,which inhibit tumor cell growth and play an important role in gene regulation.Moreover,some molecules that can bind to G4s and stabilize G4s structure may become potential drugs for gene therapy.Therefore,understanding the binding mode and interaction details between G4s and its ligand is very important for drug design,but still remain great challenges.In this work,three ligands,including fangchinoline(FAN),epiberberine(EPI)and hemin,which specifically interact with G4 through different binding modes,were selected to evaluate the feasibility and the potential of our SERS method in analyzing G4-ligand complexes.The results showed that we could obtain the effect of ligands on the structural change of G4s through the significant changes in anti/syn glycosidic angle conformations,loop shape,hydrogen bonds,and stacking interactions.This information is significantly helpful in understanding the specificity of G4-ligand complexes,and strongly suggests that the SERS method could become a powerful tool for screening specific G4-binding drugs in a fast,simple,low cost and accurate way.This detailed information regarding G4-ligand interactions is expected to enhance understanding of the molecular mechanisms of mutagenic activity.