Xin Huang

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36xa0% brighter than Spinach when expressed in E.xa0coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore

Polymerase chain reaction coupled with signal generation offers sensitive recognition of target DNA sequence; however, these procedures require fluorophore-labeled oligonucleotide probes and high-tech equipment to achieve high specificity. Therefore, intensive research has been conducted to develop reliable, convenient, and economical DNA detection methods. The relay PCR described here is the first sequence-specific detection method using a small-molecule fluorophore as a sensor and combines the classic 5-3 exonuclease activity of Taq polymerase with an RNA mimic of GFP to build a label-free DNA detection platform. Primarily, Taq polymerase cleaves the 5 noncomplementary overhang of the target specific probe during extension of the leading primer to release a relay oligo to initiate tandem PCR of the reporting template, which encodes the sequence of RNA aptamer. Afterward, the PCR product is transcribed to mRNA, which could generate a fluorescent signal in the presence of corresponding fluorophore. In addition to high sensitivity and specificity, the flexibility of choosing different fluorescent reporting signals makes this method versatile in either single or multiple target detection.

Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin.

Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

Colorimetric PCR-Based microRNA Detection Method Based on Small Organic Dye and Single Enzyme

microRNAs (miRNAs) have been a class of promising disease diagnostic biomarkers and therapeutic targets for their important biological functions. However, because of the high homology, interference from precursors (pri-miRNA, pre-miRNA), as well as limitations in the current assay technologies, it poses high demand and challenge for a specific, efficient, and economic miRNA assay method. Here, we propose a new miRNA detection method based on a label-free probe and a small organic dye with sequence dependence, realizing the sequence-specific and colorimetric detection of target miRNA. What is pleasantly surprising, only one enzyme is enough to propel the whole miRNA assay process, greatly simplifying the reaction component and detection process. Together with PCR amplification for the high enough sensitivity and three checks for specificity control, a detection limit of 5 fM was obtained and even one mutation could be discriminated visually. Overall, the new method makes much progress in convenience and economy of PCR-based miRNA assay method so that miRNA assay is going to be more friendly and affordable.

Weigh Biomaterials by Quantifying Species-specific DNA with Real-time PCR

What’s on the label is not what’s in the bottle, from food products to herbal medicinal products (HMPs), economically-motivated biomaterials adulteration is a long-term problem affecting the food and drug industry. Accurate identification of the biomaterial ingredients in processed commodities is highly desirable. In this field, DNA-based techniques have proved to be powerful tools to overcome qualitative challenges. However, is it possible to quantify the weight of biological materials with PCR? Therefore, a basic scientific question needs to be answered: what’s the relationship between DNA content and the mass of biological materials? Is DNA content directly proportional to the mass of biological materials as most of the researchers previously thought? In this study, we firstly found that there exists a linear relation between DNA contents and the weight of biomaterials indeed when the analytical practices are fully controlled. In this case, the mass of targeted biomaterials in the highly processed commercial products can also be calculated by quantifying the species-specific DNA through classic real-time PCR with a good reproducibility.

A general solution for opening double-stranded DNA for isothermal amplification

Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it.