Hanping He

Application of nanomaterials in the bioanalytical detection of disease-related genes.

In the diagnosis of genetic diseases and disorders, nanomaterials-based gene detection systems have significant advantages over conventional diagnostic systems in terms of simplicity, sensitivity, specificity, and portability. In this review, we describe the application of nanomaterials for disease-related genes detection in different methods excluding PCR-related method, such as colorimetry, fluorescence-based methods, electrochemistry, microarray methods, surface-enhanced Raman spectroscopy (SERS), quartz crystal microbalance (QCM) methods, and dynamic light scattering (DLS). The most commonly used nanomaterials are gold, silver, carbon and semiconducting nanoparticles. Various nanomaterials-based gene detection methods are introduced, their respective advantages are discussed, and selected examples are provided to illustrate the properties of these nanomaterials and their emerging applications for the detection of specific nucleic acid sequences.

A Small Molecule Affecting the Replication of Trinucleotide Repeat d(GAA)n

A newly designed ligand, methylcarbamoylnaphthyridine dimer (MCND), was synthesized and characterized. Ligand binding to d(GAA)(10) was investigated by UV thermal denaturation, circular dichroism spectroscopy, surface plasmon resonance, and cold-spray-ionization time-of-flight mass spectrometry. The results indicated that MCND bound to the d(GAA)(n) repeat to form a stable hairpin structure with a major binding stoichiometry of 3:1. The most likely binding site was identified as the G-G mismatch in the AGA/AGA triad. The polymerase stop assay showed that MCND binding to the d(GAA)(n) repeat effectively interfered with the extension of the primer at the first two GAA sites on the template with both prokaryotic Taq DNA polymerase and human DNA polymerase alpha.

A small molecule regulates hairpin structures in d(CGG) trinucleotide repeats

Unusual expansion of trinucleotide repeats has been identified as a common mechanism of hereditary neurodegenerative diseases. Although the actual mechanism of repeat expansion remains uncertain, trinucleotide repeat instability may be related to the increased stability of an alternative DNA hairpin structure formed in the repeat sequences. Here we report that a synthetic ligand naphthyridine carbamate dimer (NCD) selectively bound to and stabilized an intra-stranded hairpin structure in CGG repeat sequences. The NCD-CGG hairpin complex was a stable structure that efficiently interfered with DNA replication by Taq DNA polymerase. Considering the sequence preference of NCD, the use of NCD would be valuable to investigate the genetic instabilities of CGG/CCG repeat sequences in human genomes.

Facile electrochemical biosensor based on a new bifunctional probe for label-free detection of CGG trinucleotide repeat

We have developed a simple and label-free electrochemical assay to detect CGG trinucleotide repeat. For this purpose, a new bifunctional probe (FecNCD2) was developed, in which a recognition part (naphthyridine carbamate dimmer, NCD) was connected with an electro-active part (ferrocenyl group) using a chain of -CO-NH-CH2-CH2-. The results of circular dichroismic measurements indicated that FecNCD2 exhibited a superior performance for selective binding to CGG trinucleotide repeats compared to a previous bifunctional electrochemical probe connected with shorter linker -CH2- (FecNCD1). Then, the electrochemical properties of FecNCD2 were evaluated and were found to show a good redox response due to the ferrocene moiety. Owing to the high performances of FecNCD2, the label-free electrochemical biosensor for CGG repeats was constructed by immobilizing them onto gold disk electrode and by using FecNCD2 as an electrochemical probe in solution. Further CGG repeats in solution were confirmed to be detectable using the CGG modified biosensor in competitive experiments, i.e., by treating it in test solutions containing FecNCD2 and d(CGG)10 or others. No interference of ct-DNA on the CGG detection was also confirmed with this approach. The strategy should have significant potential for the development of versatile and low-cost biosensor for early diagnosis and treatment of neurodegenerative diseases associated with trinucleotide repeats.

Small molecule modulates hairpin structures in CAG trinucleotide repeats.

Short tandem DNA repeats are widely observed from prokaryotic to eukaryotic genomes. In humans, unusual expansions of trinucleotide repeats cause severe neurodegenerative diseases. For examples, the d(CAG)n expansion mutation in the first exon of the huntingtin gene causes Huntington’s disease. Fragile X syndrome is caused by an expansion of the d(CGG)n repeat in the coding-sequence of the FMR1 (fragile X mental retardation 1) gene. Several cellular processes involving DNA replication, repair, and recombination are related to the instability of repeat sequences. Most mechanisms responsible for repeat instability assume a formation of unusual non-B DNA conformations, such as hairpins, triplexes, and quadruplexes in a parent or daughter strand during DNA synthesis. Consequently, an extension of the slipped structure would produce a repeat expansion or contraction in repeat tracts, although the actual mechanism still remains uncertain. Small ligands that can bind and stabilize particular structures in the repeat regions would help our understanding of an abnormal expansion of repeats at the molecular level. We have recently reported that a synthetic ligand, naphthyridine-azaquinolone (NA), stabilizes CAG/CAG-triad containing DNA (Scheme 1 A). Structural characteristics have revealed that the naphthyridine and azaquinolone moieties in NA exhibit complementary hydrogen bonding to guanine and adenine, respectively, and make two cytosine bases flip out from the CAG/CAG triad (Scheme 1 B). These features explain the remarkable selectivity of NA against the CAG/CAG triad. Here, we demonstrate that NA can induce hairpin secondary structures on d(CAG)n repeats, which then efficiently interfere with DNA replication by Taq DNA polymerase. First, the biophysical and biochemical properties of a d(CAG)10, 30-mer ten-repeats of CAG trinucleotides, were investigated by circular dichroism (CD) and UV analysis (Figure 1). In the absence of NA, d(CAG)10 showed a positive broad signal at around 270 nm and a negative signal at 255 nm (Figure 1 A). Upon addition of NA, distinct spectral changes depending on NA concentration were observed by a negative induced CD at around 320 nm and a strong positive peak at 250 nm. No significant spectral change was observed in d(CTG)10 repeats even at 50 mm NA (Figure S1 in the Supporting Information), which indicated that NA preferentially bound to a repetitive CAG strand. UV thermal denaturation studies showed that the thermal stability of the d(CAG)10 was largely enhanced with a concomitant increasing concentration of NA (Figure 1 B). As NA concentration increased, the melting temperature of NA-induced hairpins rose from 47.1( 0.7) 8C to 78.8( 0.1) 8C at 50 mm NA, surpassing the melting temperature of d(CTG)10 hairpins (54.4 0.3 8C; Figure S2 in the Supporting Information). Taken together, NA could modulate the stability of the CAG hairpins without affecting its complementary CTG hairpins. To further characterize NA-bound structures in CAG repeat sequences, a polymerase-stop assay was performed on the DNA templates containing repeat sequences (Figure 2 A). Stabilization of hairpin secondary structures in repeat sequences resulted in the production of truncated complementary DNA products due to the inhibition of DNA synthesis, which was detected by PAGE analysis. In the absence of NA, the 20-mer primer that hybridized to the 3’-end of the template was fully elongated by Taq DNA polymerase in the d(CAG)10-containing templates; this suggests that no stable structures were formed that efficiently inhibited DNA synthesis. In contrast, addition of NA effectively inhibited DNA elongation to produce distinct truncated products in a concentration-dependent manner in relation to NA (Figure 2 B); this suggests that NA-induced hairpins were stable enough to efficiently interfere with DNA synthesis by Taq polymerase. Selective binding of NA to CAG repeats was assessed by comparison with DNA templates conScheme 1. A) Hydrogen bonding between naphthyridine-azaquinolone (NA) and G–A mismatch. B) Schematic illustration of the NA–CAG/CAG triad complex confirmed by NMR spectroscopy. Black rectangles: 2-amino-1,8-naphthyridine moiety; gray rectangles: 8-azaquinolone moiety.

A novel ratiometric fluorescent probe for selective detection of bisulfite in living cells

A new type of ratiometric fluorescent probe, 8-(diethylamino)-2-methyl-4-oxo-4H-pyrano[2,3-b]chromen-10-ylium tetrafluoroborate (APCT), is reported. A new type of ratiometric fluorescent probe, 8-(diethylamino)-2-methyl-4-oxo-4H-pyrano[2,3-b]chromen-10-ylium tetrafluoroborate (APCT), is reported. This probe can successfully detect bisulfite anions with a detection limit of 6.1 × 10−7 M based on Michael-type addition reaction in pH = 7.4 Tris buffer solution containing 70% DMSO (v/v). The response time of the probe is approximately 5 min. Upon treating with bisulfite anions, the probe exhibits an obvious blue shift from 560 nm to 510 nm and the fluorescence intensity ratio at 510 nm and 560 nm (I510/I560) displays a good linear relationship with the concentration of HSO3− in the range of 20–120 μM. Additionally, the preliminary cell imaging experiments using HeLa cells demonstrate that the probe can be used to detect intracellular bisulfite anions.

Surface protein imprinted magnetic nanoparticles for specific recognition of bovine hemoglobin

Molecular imprinting for the detection of protein has gained great interest in recent years. For this purpose, we prepared magnetic molecularly imprinted polymers (MIPs) for the recognition of bovine hemoglobin (BHb) through the surface imprinting technique with two-stage core–sell sol–gel polymerization on the surface of silica modified Fe3O4 nanospheres. 3-Aminopropyltriethoxylsilane and octyltrimethoxysilane were chosen as monomers to construct the MIP layer. The morphology and structure property of prepared nanoparticles were characterized by TEM, X-ray diffraction, Fourier transform infrared spectrometry, and the vibrating sample magnetometer. The obtained magnetic MIPs with high saturation magnetization (60 emu g−1) made it easy to separate the target protein from solution by an external magnetic field. The adsorption and recognition performance of this magnetic MIPs was discussed through adsorption kinetics, adsorption isotherms, special selectivity, reusability and reproducibility tests. It turned out that the magnetic MIPs showed a relatively high adsorption capacity of 124.86 mg g−1 and excellent selectivity towards BHb with a separation factor of 1.99. Moreover, the adsorption capacity of magnetic MIPs was not significantly reduced after three continuous adsorption and elution processes, which indicated their good reusability for at least three repeated cycles.

DNA Labeling by Ligand Inducible Secondary Structure

Since the human genome sequence has been determined, detection of the nucleotide base at the site of single nucleotide polymorphisms (SNPs) is one of the topics of current chemical research. Simple, accurate, and cost effective methods of SNP typing would be necessary for personalized medicine. Towards this end, a number of SNP typing methods have been investigated and reported. Most of these methods required an allele-specific oligonucleotide (ASO) as a probe, which was modified by fluorescent dyes and/or other chemicals as a ACHTUNGTRENNUNGreporting tag. A drawback of the use of ASO probes was the necessity for the discrimination or separation of a fully matched duplex from a singly mismatched duplex produced by hybridization of ASO probes to the target DNA. In addition, the ASO probes were mostly used after PCR amplification of the DNA samples being tested. The separation of the target strand from the duplex or selective amplification of the target strand by asymmetric PCR was necessary for the effective hybridization of ASO probes. Thus, SNP typing methods that are operational during the amplification process would be more favorable than those postamplification methods from the viewpoint of a simple analytical procedure. On the basis of the accumulated knowledge on SNP typing, the next chemical challenge concerning SNP typing is to propose truly practical methods that are applicable to SNP in any sequence. We report herein, our chemical approach to practical SNP typing based on allele-specific PCR integrated with a new concept of DNA-labeling by ligand-inducible secondary structure (LISS). PCR is currently one of the most fundamental technologies in biology. Besides the original purpose of amplifying DNA fragments, PCR provides information regarding the presence or absence of a particular DNA sequence in the sample DNA. PCR is recognized as an important tool for reliable diagnosis of infectious diseases and detection of genetic variations. 5] Allele-specific PCR (AS-PCR) detects point mutations depending on a match or mismatch base pair between the 3’ terminus of the primer and the template sequence. Allele specificity in PCR would rely on the high fidelity of DNA polymerase in addition to improved primer design. Methods detecting the double stranded DNA produced, pyrophosphate, and a signal and/or fragments produced from reporter DNA probes have been studied for monitoring PCR progress. We have investigated a new concept for labeling PCR primers by LISS to monitor the progress of PCR by detecting the amount of the PCR primer, which would be decreasing as the PCR progresses. The chemical basis of LISS is selective ligand binding to the single stranded DNA, resulting in a large structural change of the DNA. The requirement of structural change on ssDNA reduced the chance of ligand binding to dsDNA, for ACHTUNGTRENNUNGexample, PCR products. The PCR primer was labeled at the 5’-end with a short single stranded tag of the trinucleotide repeat sequence (TRS-tag). We have examined dACHTUNGTRENNUNG(TGG)n as the TRS-tag for labeling the PCR primer, because 1) a single stranded d ACHTUNGTRENNUNG(TGG)n sequence does not have stable secondary struc-

HBT-based turn-on fluorescent probe for discrimination of homocysteine from glutathione/cysteine and its bioimaging applications

Based on ESIPT and heavy atom effect strategy, we firstly reported a new type of turn-on fluorescent probe for Hcy. This probe displayed fast response time (15 min), excellent selectivity over other interfering species and high sensitivity with a low detection limit of 1.6 × 10−7 M in PBS buffer containing 1% CH3CN (v/v). Moreover, the probe was also successfully applied for fluorescence imaging of Hcy in HeLa cells.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine bound to the A-A mismatch in the CAG/CAG base triad in dsRNA.

A dimeric form of N-methoxycarbonyl-2-amino-1,8-naphthyridine (MCND) connected at the C2 position with a three-atom linker was examined for the binding to mismatches in double stranded RNA. Despite the fully complementary hydrogen bonding groups to guanine, MCND did not bind to guanine-guanine mismatch but did to adenine-adenine mismatch. The base pairs flanking the mismatch had weak effect on the binding, with showing the strongest binding to the A-A mismatch in the CAG/CAG sequence. The A-A mismatch in the GAC/GAC sequence was a poor substrate for the MCND binding. A monomeric derivative of MCND and another derivative lacking a methylcarbamate group showed negligilble binding to the A-A mismatch and the sequence selectivity. These results are important clues for the better molecular design of RNA binding small molecules.