Preliminary Study On The Effect And Mechanism Of Riboflavin Photochemical Treatment On Pathogen Nucleic Acid

Background Blood transfusion is an indispensable means of modern medical treatment,but new and recurrent menstrual blood-borne pathogens still threaten the safety of blood transfusion.Blood pathogen inactivation technology is a promising method to further reduce the risk of transfusion infection.It can be used as the last barrier to ensure blood safety.As a commonly used photosensitizer,the safety of riboflavin has been fully proven.Compared with other pathogen inactivation technologies,riboflavin photochemical technology does not need to remove excess riboflavin,thus simplifying the processing process and reducing the loss of blood products.Therefore,more potential of photochemical pathogen inactivation of riboflavin can be realized through further research and development.Riboflavin has been found to be a 3-loop planar structure that binds to nucleic acids and is intercalated between DNA and RNA bases.When cross-linked riboflavin is activated by ultraviolet or visible light,the guanosine base is oxidized,resulting in a single strand break in the nucleic acid.In this process,the damage mechanism of nucleic acid has three aspects :(1)the direct electron transfer reaction of guanosine oxidation,(2)the generation of oxygen radical,(3)the generation of hydrogen peroxide radical and hydroxyl radical.The three absorption peaks of riboflavin are in the UV region at 221 nm,265nm and375 nm respectively,and there is one absorption peak in the visible region at 446 nm.Therefore,both visible and ultraviolet light can activate riboflavin.The second damage mechanism of riboflavin to produce oxygen free radicals is the focus of this research.It has been known that riboflavin can produce a large amount of singlet oxygen under ultraviolet light,it has been found in photodynamics that the photodynamic action of singlet oxygen can kill cells.Therefore,riboflavin and singlet oxygen produced can also be linked together in photochemical reactions to carry out more in-depth research on it.This experimental platform has established the exploration of experimental conditions for riboflavin photochemical method in the previous work.On this basis,this project will optimize the conditions for the establishment of plasma riboflavin photochemical pathogen inactivation system,and carry out preliminary exploration on the mechanism of riboflavin photochemical method acting on viral nucleic acid.Objective The mechanism of photochemical inactivation of plasma pathogens by riboflavin was investigated.Methods1.By changing the experimental conditions of riboflavin photochemical method,the optimal inactivation conditions were optimized,mainly aiming at the influence of temperature,riboflavin concentration and ultraviolet illumination time on the effect;2.Qsep100 nucleic acid protein analyzer was used to detect the integrity of RNA,and large fragment step-step PCR was used to detect the damage to the whole gene of the virus by riboflavin photochemical method.The possible damage sites of the virus were initially located,and the nucleic acid base sequence was analyzed;3.Change the conditions to detect whether singlet oxygen content has changed,and then use singlet oxygen scavenger to remove singlet oxygen generated in the experiment,and then compare the experimental inactivation effects to explore whether singlet oxygen may participate in pathogen inactivation.Results1.Optimized conditions can be established: UVB light source riboflavin pathogen inactivation system with riboflavin concentration of 50μmol/L,illumination time of20 min and temperature of 20℃.2.By Qsep100 nucleic acid protein analyzer,it was found that the degree of fracture of inactivated viral nucleic acid was much higher than that of non-inactivated viral nucleic acid,and the degree of fragmentation of viral nucleic acid was increased after inactivated.The destruction site was not sequence specific but related to the base proportion.PCR results showed that the fragment bands showed a gradual weakening trend,and the bands at 2726-3697 bp of the fourth fragment were significantly weakened,followed by the bands at 5449-6236 bp of the seventh fragment,indicating that riboflavin-mediated nucleic acid breakage is a continuous process,which is related to the sensitivity of each gene fragment.3.In the detection of singlet oxygen content,it was shown that the longer the light time and the higher the temperature,the more singlet oxygen production,the better the inactivation effect,which was positively correlated with the inactivation conditions of pathogens by riboflavin photochemical method,and the inactivation effect greatly decreased after the use of active oxygen scavenging.Studies showed that singlet oxygen involved in the inactivation process affected the inactivation effect.Conclusion1.In this study,the optimized conditions for photochemical inactivation of pathogens by riboflavin were established as follows: riboflavin concentration of 50μmol/L,light time of 20 min and temperature of 25℃.2.The mechanism of riboflavin photochemical inactivation of virus has no sequence specificity of nucleic acid but has a tendency of base specificity.Related to purine content ratio,riboflavin photochemical inactivation of virus specifically mediates the break of nucleic acid sequence and prevents subsequent replication.3.Singlet oxygen produced by riboflavin photochemical method is one of the key factors affecting the inactivation effect,and the experimental results show that the content of singlet oxygen is positively correlated with the inactivation effect.