Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA

Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.

Graphene oxide and molecular beacons-based multiplexed DNA detection by synchronous fluorescence analysis

We have developed a multiplexed DNA detection method based on graphene oxide （GO） and molecular beacons （MBs） by synchronous fluorescence analysis, demonstrated it by an oligonucleotide sequence of wild-type HBV （T1） and a re- verse-transcription oligonucleotide sequence of the RNA fragment of HIV （T2） as a model system. In the absence of targets DNA, FAM-tagged free MB probes （PHBv） and ROX-tagged free MB probes （PHIv） are adsorbed on GO via π-π interactions between DNA nucleobases and nucleosides, and the π-rich GO brings the fluorophores of MB and GO into close proximity. And then, the fluorescence of fluorophores is quenched by GO. But in the presence of targets DNA, PHBV and PHW hybridize with their targets DNA resulting in the formation of double-stranded DNA （dsDNA）, causing the separation of PHBV and PHW from the surface of GO and the recovery of the fluorescence of fluorophores （FAM and ROX） simultaneously. The simultane- ous detection of T1 and T2 can be realized by measuring fluorescence signals of FAM and ROX, respectively. Under the op- timum conditions, the fluorescence intensities of two dyes all exhibit good linear dependence on their target DNA concentra- tion in the range of 5×10-11-5×10 9 M. The detection limit of T1 is 3×10-11 M （3σ）, and that of T2 is 2×10-11 M. Compared with other methods for DNA detection based on GO, the proposed method has some advantages including higher selectivity and shorter analytical time.