Single Molecule Fluorescence Study The Stepping Kinetics Of Pif1 Helicases And Method Expansion

Pif1 helicase is a kind of DNA molecular motor,which obtains energy by combining and hydrolyzing ATP.Pif1 can conduct directional translocation in the direction of 5’to 3’on single strand DNA,and drive many physiological functions at the same time.Pif1 is distributed in all eukaryotes and some prokaryotes.Pif1 is not only a multifunctional helicase,but also a multi-domain helicase.In addition to the relatively conservative helicase core domain of the Pif1 helicase family,some Pif1also has two accessory domains namely the C-terminal domain and the N-terminal domain.For example,Saccharomyces cerevisiae Pif1（Sc Pif1）has a C-terminal domain and an N-terminal domain.Due to the difficulty in analyzing the full length Pif1 structure,there is currently insufficient research on the accessory structural domains of Pif1.FRET is a commonly used single molecule fluorescence technique,which is widely used in the study of helicase step kinetics.The DNA used in previous studies of helicase has a forked structure and can only achieve a resolution of 1 bp.In this paper,the resolution of 0.5 bp can be achieved by using DNA nanotensioner to enhance FRET.In this study we found that Pif1 has two kinds of unwinding modes,one is random length unwinding and the other is critical length unwinding.By mutating Pif1,we found that the C-terminal domain of Pif1 can regulate the unwinding modes of Sc Pif1.In addition,the DNA nanotensioner constructed in this paper can achieve a resolution of one nucleotide,and the mechanism of the C-terminal domain regulating the unwinding modes of Pif1 was revealed by this DNA nanotensioner.The findings of this paper make up for the shortcomings of study Pif1by structural biology and provide new insights into the structure and functional relationship of Pif1.FRET can measure the distance change between the fluorescent donor and the fluorescent receptor,and it can obtain one-dimensional distance information.However,FRET also has some limitations in studying molecular motor or biological macromolecules due to it’s difficult to measure the height change of fluorescent label.In recent years,SIFA technology based on the FRET principle has developed using two-dimensional materials as dielectric acceptors,which can detect the height changes of fluorescent donors from the dielectric surface.In recent years,SIFA based on graphene and graphene oxide has been applied to study the normal movement and insertion behavior of membrane proteins on the surface of cell membranes.The detection range of SIFA is limited by the characteristic quenching distance d₀of the medium material.The existing SIFA technology based on graphene and graphene oxide has undetectable area.The d₀of graphene oxide is 4 nm,and that of graphene is18 nm.The preparation of two-dimensional materials which has a suitable d₀between graphene and graphene oxide face difficulties in material selection and preparation.In addition,a single two-dimensional material has a fixed d₀thus the detection range is limited.Therefore,there is an urgent need to develop controllable d₀materials for SIFA.In this paper,graphene oxide is reduced by thermal reduction method,which can rapidly and cheaply prepare reduced graphene oxide and be used in SIFA technology.The d₀of reduced graphene oxide can be dynamically adjusted by controlling the temperature of thermal reduction.Furthermore,we takes reduced graphene oxide as the medium receptor,and uses alternating laser to excite the fluorescence donor and receptor alternately,realizing the synchronous measurement of the distance between the fluorescence donor and the receptor,as well as their respective distance from the surface of reduced graphene oxide,and realizing the combination of SIFA and FRET technology.The SIFA-FRET technology developed in this paper can select appropriate fluorescent pairs and reduce graphene oxide according to the experimental requirements.the sensitive area for detection in three-dimensional space can be flexibly selected.SIFA-FRET has broad application prospects in studying the position and conformational transformation of biological macromolecules in three-dimensional space.