Xianjin Xiao

Nucleic acid fluorescent probes for biological sensing.

Nucleic acid fluorescent probes are playing increasingly important roles in biological sensing in recent years. In addition to the conventional functions of single-stranded DNA/RNA to hybridize with their complementary strands, affinity nucleic acids (aptamers) with specific target binding properties have also been developed, which has greatly broadened the application of nucleic acid fluorescent probes to the detection of a large variety of analytes, including small molecules, proteins, ions, and even whole cells. Another chemical property of nucleic acids is to act as substrates for various nucleic acid enzymes. This property can be utilized not only to detect those enzymes and screen their inhibitors, but also employed to develop effective signal amplification systems, which implies extensive applications. This review mainly covers the biosensing methods based on the above three types of nucleic acid fluorescent probes. The most widely used intensity-based biosensing assays are covered first, including nucleic acid probe-based signal amplification methods. Then fluorescence lifetime, fluorescence anisotropy, and fluorescence correlation spectroscopy assays are introduced, respectively. As a rapidly developing field, fluorescence imaging approaches are also briefly summarized.

A universal mismatch-directed signal amplification platform for ultra-selective and sensitive DNA detection under mild isothermal conditions

We describe a novel mismatch-directed signal amplification method which can be performed easily under mild isothermal conditions for the ultra-selective and sensitive detection of any DNA sequence of interest. By taking advantage of the unique discrimination properties of an endonuclease IV and lambda exonuclease coupling system in cleavage reactions of DNA duplexes containing apurinic/apyrimidinic sites with different adjacent mismatched bases, the established assay enables sensitive detection of 1.0 fmol of target strand and selective differentiation of 0.5% target strand from single-base different sequences at 37 °C without the need for temperature adjustment.

Endonuclease IV discriminates mismatches next to the apurinic/apyrimidinic site in DNA strands: constructing DNA sensing platforms with extremely high selectivity

A unique capability of Endonuclease IV in discrimination of mismatches neighboring a natural abasic site in DNA strands has been demonstrated, which enables genotyping of SNPs with high discrimination factors and differentiation of as low as 0.1-0.01% of target DNA strands from a large background of single-base different interfering strands.

A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations

Abstract Genetic mutations are important biomarkers for cancer diagnostics and surveillance. Preferably, the methods for mutation detection should be straightforward, highly specific and sensitive to low-level mutations within various sequence contexts, fast and applicable at room-temperature. Though some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a branch-migration based fluorescent probe (BM probe) which is able to identify the presence of known or unknown single-base variations at abundances down to 0.3%-1% within 5 min, even in highly GC-rich sequence regions. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 89–311 by measurement of their respective branch-migration products via polymerase elongation reactions. The BM probe not only enabled sensitive detection of two types of EGFR-associated point mutations located in GC-rich regions, but also successfully identified the BRAF V600E mutation in the serum from a thyroid cancer patient which could not be detected by the conventional sequencing method. The new method would be an ideal choice for high-throughput in vitro diagnostics and precise clinical treatment.

New advances in molecular recognition based on biomolecular scaffolds.

In this review, we summarize recent advances in the development of molecular recognition components based on the biomolecular scaffolds of proteins and nucleic acids for specific recognition of miscellaneous targets. In addition to the widely adopted recombinant antibody fragments, designed ankyrin repeat proteins and modular peptide repeats of transcription-activator-like effectors for base-specific recognition of DNA sequence are also briefly introduced. For the nucleic acid based molecular recognition systems, aptamers, including slow off-rate modified aptamers, DNAzymes, and synthetic DNA-like oligomers for versatile biorecognition are described. Finally, we discuss the remaining challenges and future research directions in the field.

Combination of a modified block PCR and endonuclease IV-based signal amplification system for ultra-sensitive detection of low-abundance point mutations.

By combination of a modified block PCR and endonuclease IV-based signal amplification system, we have developed a novel approach for ultra-sensitive detection of point mutations. The method can effectively identify mutant target sequence immersed in a large background of wild-type sequences with abundance down to 0.03% (for C→A) and 0.005% (for C→G). This sensitivity is among the highest in comparison with other existing approaches and the operating procedures are simple and time saving. The method holds great potential for future application in clinical diagnosis and biomedical research.