Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.

The template-directed synthesis of RNAp layed an important role in the transition from prebiotic chemistry to the beginnings of RNAb ased life,b ut the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with as et of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of ap rimer/ template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'hydroxyl occurs prior to the rate limiting step,the attackofthe alkoxide on the activated phosphate of the incoming nucleotide.T he analogs with a 3 Er ibose conformation show the fastest formation of 3'-5' phosphodiester bonds.Among those derivatives,t he reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs.t ransition states.

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