Highly Sensitive Detection Of SARS-CoV-2 RNA And N~6-methyladenosine

Since the end of 2019,corona virus disease-2019(COVID-19)has swept over 200 countries and infected more than 500 million people,which causes huge loss of life and property to all human beings.It is called the public health emergency of international concern(PHEIC)by the World Health Organization(WHO).The pathogen of COVID-19 is an RNA virus,named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Since RNA is the genetic material of SARS-CoV-2,developing a highly sensitive detection method for SARS-CoV-2 RNA is of great significance to the early diagnosis and epidemic prevention and control of COVID19.N6-methyladenosine(m6A)is formed by the methylation of the sixth N atom of adenosine A.It is the most prevalent and abundant modification in the mRNA of eukaryotes,especially higher eukaryotes.The methylation process of m6A is revisable and dynamically regulated.Adenosine A forms m6A under the catalysis of methyltransferase complex(METTL 3,METTL 14 and WTAP),and m6A can also be demethylated to adenosine A under the catalysis of the demethylase(FTO or ALKBH5).Studies have shown that m6A plays an important role in gene expression,including mRNA translation,splicing and degradation.In addition,aberrant m6A modification level is associated with various diseases(mental disorders,metabolic syndrome and cardiovascular disease)and cancers(acute myeloid leukemia,brain tumors and reproductive system-related cancers).Therefore,precisely quantitative analysis of m6A in the RNA is of great significance to m6A biological function research and diagnosis of related diseases.In this paper,we have developed a single-molecule digital counting method for quantification of SARS-CoV-2 RNA based on electric field-propelled fluorescent micromotors.Besides,by combining the m6A sensitive enzymes(T3 DNA ligase,Bst DNA polymerase,VMC10 deoxyribozyme)with nucleic acid amplification reaction or CRISPR/Casl2a system,we have established two methods to quantify m6A with high sensitivity and selectivity.(1)We have developed a single-molecule digital counting method for quantification of SARS-CoV-2 RNA based on electric field-propelled fluorescent micromotors.This method took the advantages of the peptide nucleic acids probes’high affinity and selectivity and used PNA-functionalized fluorescent microbeads to capture the SARS-CoV-2 RNA.By regulating the number of fluorescent microbeads,it achieved that one SARS-CoV-2 RNA molecule can bind to one fluorescent microbead to form a micromotor and thus established a one-by-one relationship between the SARS-CoV-2 RNA and the fluorescent microbeads.Subsequently,one SARS-CoV-2 RNA can drive one micromotor to the target district and be separated from the background under the applied electric field.Finally,digital quantification of SARS-CoV-2 RNA can be realized by counting the micromotors.This approach successfully achieved digital quantifying of SARSCoV-2 RNA at the single-molecule level and can also discriminate single-base mutation in the SARS-CoV-2 RNA.Therefore,this method has great potential in virus traceability and clinical diagnosis.(2)M6A can hinder the reverse transcriptional activity of Bst DNV polymerase and the ligation activity of T3 DNA ligase.Based on this,we developed a gap filling ligation-based rolling circle-loop mediated isothermal amplification(GLR-LAMP)method for quantifying m6A in the RNA.GLR-LAMP utilizes a specially designed padlock probe to hybridize to the target RNA and leave a single nucleotide gap at the target site.When the target site is A,the 3’ end of the padlock probe will elongate a single-base U under the catalysis of Bst DNA polymerase and then be ligated by T3 DNA ligase to form a circular DNA.When the target site is m6A,m6A will hinder the elongation of the 3’ end of the padlock probe.Due to the gap between the padlock probe’s 5’ and 3’ ends,it cannot be ligated by T3 DNA ligase to form the circular DNA.Even if the hindering efficiency of the Bst DNA polymerase is not 100%and a small number of padlock probes elongated a single-base U,m6A can also hinder the ligation of T3 DNA ligase.Thus,the amount of circular DNA products will be greatly reduced.Afterward,the circular DNA can be amplified by the rolling circle amplification and loop-mediated isothermal amplification.During the amplification reaction,SYBR Green I is used as the fluorescent dye to monitor the amplification products in real time.Finally,the m6A modification fractions can be calculated according to the reduction of the amplification products.By using Bst DNA polymerase and T3 DNA ligase to double identify the m6A,GLR-LAMP can clearly discriminate m6A from unmodified A and the selectivity is up to 100-fold.In addition,rolling circle amplification and loop-mediated isothermal amplification ensure the sensitivity of this method and as low as 1fM target RNA can be accurately detected.It was successfully applied to quantify m6A modification fractions in the MALAT1 lncRNA 2577th site from HeLa and HEK293T cells.(3)Deoxyribozyme VMC10 can specifically recognize and cleave the unmethylated A at the "GAC" site in the RNA.Based on this,we developed a deoxyribozyme mediated CRISPR/Cas 12a platform(DCAS)to quantify m6A at the specific site in the RNA.This method utilizes deoxyribozyme VMC10 to hybridize with the target RNA at the "GAC" site.It can cleave the unmethylated A with high and robust efficiency,whereas it cannot cleave m6A.After the cleavage,only the m6A-containing RNA can be amplified by RT-PCR.Subsequently,the PCR products can serve as substrates to be recognized by CRISPR/Casl2a system and then activate the trans-cleavage activity of Cas12a to cleave the reporter and generate the fluorescent signal.By monitoring the fluorescent signal intensity and comparing it with the control(without VMC10 cleavage treatment),it can directly quantify the m6A modification fractions at the target site.In addition,this method has high sensitivity.It can detect m6A-containing RNA targets as low as 100 aM and monitor the small changes of m6A modification fractions.