RESUMO
The parallel double-stranded DNA (dsDNA) demonstrates potential utility in molecular biology, diagnosis, therapy, and molecular assembly. However, techniques for the characterization of parallel dsDNA are limited. Here, we demonstrate that a series of intensive characteristic Raman bands of three parallel dsDNAs, which are stabilized by reverse Hoogsteen A+·A+ base pairs or hemiprotonated C+·C, G·G minor groove edge, Hoogsteen A·A base pairs, or Hoogsteen T·A, C+·G base pairs, have been observed by surface-enhanced Raman spectroscopy (SERS) when the gold nanoparticles modified by bromine and magnesium ions (Au BMNPs) were used as substrates. The featured bands can not only accurately discriminate parallel dsDNA from antiparallel one but also identify the strand orientation within dsDNA. The proposed approach will have a significant impact on DNA analysis, especially in the detection and differentiation of various DNA conformations.
Assuntos
Ouro , Nanopartículas Metálicas , Ouro/química , Análise Espectral Raman , Brometos , Nanopartículas Metálicas/química , DNA/químicaRESUMO
Surface-enhanced Raman spectroscopy (SERS) has been demonstrated as an effective method for elucidating secondary structural characteristics of DNA. However, the inherent complexity of the DNA conformation and the lack of SERS samples pose challenges for identifying numerous secondary structures. To address these issues, a synergistic method integrating machine learning with SERS was proposed so as to analyze the SERS spectra of 54 well-defined conformational oligonucleotides, namely, G-quadruplex (G4), i-motif (iM), double-strand (DS), and single-strand (SS) configurations. Principal component analysis (PCA) effectively segregated the oligonucleotides into three distinct conformational groups (G4s, iMs, and others). Furthermore, linear discriminant analysis (LDA), K-nearest neighbor (KNN), and support vector machine (SVM) approaches were utilized to improve the typing accuracy of 54 trained sequences. This enabled the correct classification of the structures of five untrained sequences, as well as the identification of the predominant conformations including G4, iM, and DS formed by two complementary G-rich and C-rich sequences in acidic and neutral pH conditions. The results of this study demonstrated the potential of the proposed methodology for rapid screening and prediction of secondary DNA conformations.
RESUMO
Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in label-free DNA detection. Owing to the limitation in chain length, it is however still challenging for SERS as a routine method to explore the intrinsic structural information on unmodified DNA. Here, we develop a universal SERS-based approach toward quantification of A/G in single-stranded DNAs (12 up to 28 bases) by introducing a novel interfacial agent, dichloromethane. DNA hybridization is successfully probed as evidenced by the typical SERS bands attributed to hydrogen bonds in a hairpin structure. More importantly, enlarged space of "hot spots" in SERS enables discrimination of single-base mutation in double-stranded DNA with 100 bases, which as a proof-of-concept study will pave a new avenue for highly sensitive DNA detection in clinical applications.
Assuntos
DNA/genética , Mutação Puntual , Análise Espectral Raman/métodos , Sequência de Bases , DNA/análise , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Indicadores e Reagentes , Cloreto de Metileno , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodosRESUMO
The direct characterization of DNA nanogels at the atomic level is desirable and of great significance, however, has been challenging because of structural complexity and the larger size of nanogels. Herein, we demonstrated a simple, sensitive and reliable SERS (Surface-enhanced Raman spectroscopy)-based approach towards direct monitoring microstructures, such as three types of nanogels crosslinked by DNA G-quadruplex, i-motif and GC duplex. The achievement is attributed to the detection of featured Raman bands corresponding to the formation of Watson-Crick and Hoogsteen hydrogen bonds as well as C·C+ base pairs. Importantly, this work reveals that the silver nanoparticles attaching on the surface of nanogels can form local 'hotspots' and produce high-quality of Raman spectra under the assistance of iodide, aluminum ions and dichloromethane, therefore, shows great potential for wide applications in accurate characterization of various DNA nanostructures.
Assuntos
Nanopartículas Metálicas , Análise Espectral Raman , DNA/química , Iodetos , Nanopartículas Metálicas/química , Nanogéis , Prata/química , Análise Espectral Raman/métodosRESUMO
Direct, label-free sequence analysis of DNA hybridization has been achieved by surface-enhanced Raman spectroscopy. In this work, aluminum-ion-aggregated and iodide-modified silver nanoparticles were used as substrates to obtain Raman spectra of the DNA strands with the same base composition but different sequences, which form random coils or various hairpin conformations. Upon DNA hybridization, reproducibly enhanced bands were easily observed, corresponding well to the formation of Watson-Crick hydrogen bonds, base ring breathing vibrations, and hairpin loops. These characteristic bands can be used to unambiguously distinguish the hairpins from the random DNA conformation. Moreover, by using the deoxyribose band (959 cm-1) as an internal standard to normalize the characteristic bands at 1703 cm-1 corresponding to the dG νC=O H bond, the guanine-cytosine base-pair contents and sequence in DNA hairpins can be accurately measured. Applying this method, a single base mutation in a functional double helix was confidently identified.