Characterization of Parallel-Stranded DNA Duplexes by Surface-Enhanced Raman Spectroscopy and Bromide-Modified Gold Nanoparticles.

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.

Revealing the specific interactions between G-quadruplexes and ligands by surface-enhanced Raman spectroscopy.

G-quadruplexes (G4s) regulate a variety of physiological functions related to diseases and life elongation. Therefore, G4 binding ligands, such as potential drugs in gene therapy or molecular probes for biosensing and bioimaging, are receiving extensive attention. However, identifying the binding modes and interaction details between G4s and their ligands is very challenging. Recently, we demonstrated that surface-enhanced Raman scattering (SERS) could quickly provide structural details of G4s. Herein, three G4 binding ligands that interact with the separated G4 in different ways are selected as models to evaluate the feasibility of SERS analysis in studying G4-ligand interactions. As a result, adequate SERS information indicating the specific interactions between the G4s and the ligand is obtained via using Ag IANPs as substrates. The results demonstrate that SERS is a powerful tool for revealing comprehensive and specific ligand-DNA interactions with advantages such as speed, simplicity, trace sample amount requirement, and compatibility with aqueous samples.

Revealing the effect of intramolecular interactions on DNA SERS detection: SERS capability for structural analysis.

Intramolecular interactions are key factors for constructing the secondary conformations of biomolecules and they are also vital for biomolecular functions. Their effect on the surface-enhanced Raman spectroscopy (SERS) spectra is also important for reliable label-free detection. The current work focuses on three GCGC-quadruplexes as model molecules for SERS studies, which contain both the G-quartet and the GCGC-quartet. Their spectra are compared with the ones of the G-quadruplex and the duplex. The present work presents the specific effect of intramolecular interactions, including various Watson-Crick and Hoogsteen hydrogen bonds as well as base stacking, on the SERS signals of closely-related secondary conformations. The overall results indicated a significant influence on the direct label-free detection of DNA molecules and the SERS capability for secondary structural analysis.

Accurate assembly and direct characterization of DNA nanogels crosslinked by G-quadruplex, i-motif and duplex with surface-enhanced Raman spectroscopy.

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.

Adenine shares the plane with G-quartet detected by surface-enhanced Raman spectroscopy.

DNA G-quadruplexes (G4s) formed by guanine(G)-rich sequences show diversity of structural topologies. The detection of structural details is of great significance for understanding of their functions and for the target drug design, but is very challenging. Herein, we demonstrate that the surface-enhanced Raman spectroscopy (SERS) via Ag IANPs as substrates is able to identify the numbers of Adenine (A) located on the G-quartet of the G4s. Eight G4s are selected for SERS studies. Besides the detection of series of characteristic bands indicating the formation of G4s, the intensity of the band represented A base ring breath (νA, ~733 cm-1) is observed particularly enhanced when there are A bases coplanar with G-quartet, and which is higher than the intensity of the band corresponding to G base ring breath (νG, ~655 cm-1). Furthermore, the band intensity ratio of νA to νG versus the ratio of the numbers of A on the plane to the sum of numbers of A and G shows very good linear relationship. Thus, based on the band intensities of νA to νG and their ratio in the SERS spectrum, the G-quadruplexes with or without a coplanar A base and numbers of A bases on the plane of G-quartet can be facilely identified. The method is simple, fast, low cost and sensitive to provide particular details of the structure in aqueous solution, therefore, implies widespread applications.

A stable uncompleted tetramolecular G-quadruplex formed by d(AGnA) under acidic condition.

In this work, the effects of terminal adenines on the formation and stability of tetramolecular G-quadruplexes (G4s) have been studied by electrospray ionization mass spectrometry (ESI-MS), UV, CD and NMR spectroscopy. Several evidences suggested that the sequences d(AGnA) (n = 4 or 5) form stable uncompleted tetramolecular G4 at acidic condition which is different from the canonical one in the neutral condition. In addition, hydrolysis of guanine has also been observed in acidic condition that may occur for unpaired strands rather than in complete G4. Thus, a new G4 topology containing incomplete G-quartet is proposed that is very stable and particularly presents in acidic ammonium ions solution. The information presented in this study provides the new insight on the polymorphism of G4s in acidic environment, which may help understand of the special role of adenines on the formation of G4s.

Label-Free and Highly Sensitive Detection of Native Proteins by Ag IANPs via Surface-Enhanced Raman Spectroscopy.

The use of silver nanoparticles (Ag NPs) as substrates to obtain satisfactory Raman spectra of native proteins is a simple and valuable but challenging process. Herein, the Ag NPs modified with aluminum and iodide ions (Ag IANPs) were introduced for Raman detection of proteins, including acidic BSA (PI 4.7), catalase (PI 5.4), ß-casein (PI 4.5), α-casein (PI 4.0), insulin (PI 5.35), basic myoglobin (PI 6.99), and lysozyme (PI 11.2). The Raman signals of all the detected proteins were significantly improved in comparison with the reported spectra obtained by using Ag NPs containing Na2SO4, I-, and Mg2+. Specifically, detection sensitivities of the acidic proteins were drastically increased. The limit of detection (LOD) of bovine serum albumin (BSA), α-casein, and ß-casein was 0.03 ng/mL. The LOD of insulin and catalase were 0.3 and 3 ng/mL, respectively. As the bands corresponding to disulfide bonds, α-helices, residues of Phe, Trp, and Tyr, and carboxyl groups were also greatly enhanced, it was easy to monitor the folding of native protein and the denaturation of protein under acidic and heated conditions. Thus, Ag IANPs as substrates open a way for surface-enhanced Raman spectroscopy (SERS) detection of proteins. Hence, the method can provide more valuable information about protein and, therefore, has the potential for wide applications.

Direct Approach toward Label-Free DNA Detection by Surface-Enhanced Raman Spectroscopy: Discrimination of a Single-Base Mutation in 50 Base-Paired Double Helixes.

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.

Base-Pair Contents and Sequences of DNA Double Helices Differentiated by Surface-Enhanced Raman Spectroscopy.

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.

5'-(CGA) n sequence-assisted pH-controlled assembly of supramolecular DNA nanostructure.

Herein, the DNA strands containing 5'-(CGA) n and consecutive guanines are used to construct supramolecular DNA nanostructures that are size-controlled by pH values. Additionally, the introduction of thymine linkers within DNA nanostructures is necessary to maintain the stability of long-sized nanostructures. This work also demonstrates a method for accurately building DNA nanostructures.

Structural Features of DNA G-Quadruplexes Revealed by Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) has been successfully used for the label-free detection of single-stranded oligonucleotides. However, the detection of complex DNA secondary structures remains a challenge. Structural features of diverse DNA G-quadruplexes were investigated via a novel SERS method. As a result, a series of highly reproducible and sensitive SERS signatures featuring the structures of G-quadruplexes were obtained. For the first time, we reported remarkably enhanced SERS bands corresponding to purine ring breathing vibrations. Moreover, we observed that by measuring the intensity of the bands corresponding to intramolecular hydrogen bonds, we could quantitatively assess the stability of the G-quadruplexes. Because no labels on DNA strands were present as the experiments were carried out in the solution, the fingerprint peaks reflect the native, internal structure of the G-quadruplexes accurately. The method here detailed provides new insights into the promising applications of diverse DNA structural studies.

Label-Free Detection of Tetramolecular i-Motifs by Surface-Enhanced Raman Spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) can probe DNA primary structure; however, the direct analysis of DNA secondary structure is still technically challenging. In this study, we developed a novel method based on introducing aluminum ion as the aggregation agent for silver nanoparticles, affording high-quality SERS signals of tetramolecular DNA. Thus, the formation of tetramolecular i-motifs was precisely analyzed by SERS signals for the first time and structural features involving glycosidic torsion, protonated cytosine, and cytosine rings were investigated. Moreover, the number of intercalated base pairs in i-motifs were quantified based on the relative intensities of the characteristic SERS bands. The proposed approach would have widespread applications in DNA analysis, especially for detecting various DNA secondary structures.

Construction of a junction DNA nanostructure and modulation of the junction switching to quadruplexes.

A junction DNA nanostructure has been successfully built in lithium acetate buffer solution at a near-neutral pH value through the connection of two slipped junction structures that are formed by G-rich and C-rich strands. The GC-rich duplex junctions in the nanostructure can be switched to G-quadruplexes and i-motifs in weakly acidic potassium acetate solution, which leads to the assembly of DNA nanostructures composed of alternating quadruplex and duplex DNA structures. The transformation between different DNA nanoarchitectures may be applied to the operation of 'DNA nanomachines'.