Xin Su

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.

A transformer of molecular beacon for sensitive and real-time detection of phosphatases with effective inhibition of the false positive signals

Molecular beacons (MBs) have shown great potential in measurement of enzyme activities. However, currently available methods for monitoring of phosphatases only use MBs as a signal reporter. Extra substrates for the phosphatases are needed to hybridize to the MB either as a primer or as a template. Moreover, few MB-based methods have been used to detect enzyme activities in real biological samples due to insufficient sensitivity or false positive interference signals caused by nonspecific nucleases. In this work, a novel type of fluorescent probe was designed and synthesized for monitoring of phosphatases by integrating the DNA substrate and the signaling structures into a single molecule. Such a new design not only significantly simplified the probing system and greatly enhanced the sensitivity, but also offered a practical way to guard against the false-positive signal problems in the application to real samples. The unique design of the assay format should be widely applicable to many other enzymatic assays using oligonucleotide fluorescent probes.

Discrimination of the false-positive signals of molecular beacons by combination of heat inactivation and using single walled carbon nanotubes.

Molecular beacons (MBs) have been extensively used for real-time monitoring of RNA/DNA and protein molecules. However, such versatility also brings about multiple sources of positive signals. Moreover, the covalently attached quencher or fluorophore may even be cleaved from the strand by the exonucleases, followed by complete degradation of the probe. These undesirable false-positive signals (FPSs) have seriously limited the application of MBs to detect real world samples. In this paper, we propose a novel and efficient approach for discrimination of FPSs of MBs due to non-specific MB-protein interactions and nuclease degradation by combination of heat inactivation and using single walled carbon nanotubes (SWNTs). The mechanisms of different DNA-protein interactions that are responsible for the generation of FPSs of MBs were investigated in detail. The proposed strategy can quickly identify the possible sources of FPSs caused by mechanisms other than hybridization in detecting real samples, which would be very helpful in choosing a proper way to modify the structure of the MBs or using a specific inhibitor. The established method was successfully applied to verify the FPSs in the measurement of a plant tissue sample.

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.