Patent ID: 12241886

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is described in conjunction with the drawings below.

The object of the invention is initiated by a hexamer cyclic helicase of bovine papillomavirus, which plays a role of assisting genomic DNA replication in bovine papillomavirus. Wild-type protein is an ATP-dependent helicase of bovine papillomavirus that exists for melting DNA of a double-helix during viral replication. It is reported that the protein binds to single-stranded DNA and attaches to the genomic DNA replication initiation site in a hexamer form at the initiation of viral genome replication. And under the condition of energy provided by atpase hydrolysis of ATP, it continues to melt along the single-stranded DNA from the replication fork in the 3′-5′ direction.

The full-length protein amino acid sequence (SEQ ID No. 5) of wild-type bovine papillomavirus double-strand DNA helicase E1, has 605 amino acids in total. The italicized and underlined part is E1-1 (306-577):

Mandkgsnwdsglgcsyllteaecesdkeneepgagvelsvesdrydsqdedfvdnasvfqgnhlevfqalekkageeqilnlkrkylgssqnssgseasetpvkrrksgakrrlfaeneanrvltplqvqgegegrqelneeqaishlhlqlyksknatvfklglfkslflcsfhditrlfkndkttnqqwvlavfglaevffeasfellkkqcsflqmqkrsheggtcavylicfntaksretvrnlmanmlnvreeclmlqppkirglsaalfwfksslspatlkhgalpewiraqttlneslqtekfdfgtmvqwaydhkyaeeskiayeyalaagsdsnaraflatnsqakhvkdcatmvrhylraetqalsmpayikarcklatgegswksiltffnyqniehtitfinalklwlkgipkknclafigppntgksmlcnslihflggsvlsfanhkshfwlasladtraaliddathacwryfdtylrnaldgypvsidrkhkaavqikappllvtsnidvqaedrylylhsrvqtfrfeqpctdesgeqpfnitdadwksffvrlwgrldlideeedseedgdsmrtftcsarntnavd.

The invention discovers that the bovine papillomavirus double-stranded DNA helicase protein and its homologous protein can be used to prepare nanopores and membranes containing conductive pore. The prepared nanopore and the membrane containing conductive pore can be used for the preparation of a single molecule sensor or kit for characterizing a target sample. It provides a new and effective choice for the field.

The optional information of the bovine papillomavirus double-stranded DNA helicase protein is shown in table 2 below:

TABLE 2Information of bovine papillomavirus double-stranded DNA helicaseproteinHomol-ogousproteinNCBI accessionSimilar mutated aminonameSourcenumberacidE1BovineNP_056739.1Compare thepapillomavirushomologous protein-1with the bovinepapillomavirusdouble-strand DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is found to be100%. Therefore, theprotein could also adoptthe following mutationscheme to improve thepore performance.E1BovineAAB35071.1Compare thereplicationpapillomavirushomologous proteinprotein-1with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is found to be100%. Therefore, theprotein could also adoptthe following mutationscheme to improve thepore performance.E1BovineAFV52373.1Compare thepapillomavirushomologous protein-1with the bovinepapillomavirusdouble-strand DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is found to be100%. Therefore, theprotein could also adoptthe following mutationscheme to improve thepore performance.E1DeltaACR78098.1Compare thepapillomavirushomologous proteingenus -4with the bovinepapillomavirusdouble-strand DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >99%, andthe above-mentionedmutant amino acidposition is same as E1-2(306-605). Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1DeltaACR78091.1Compare thepapillomavirushomologous proteingenus -4with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >98%, andthe above-mentionedmutant amino acidposition is same as E1-2(306-605). Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1DeltaACR78081.1Compare thepapillomavirushomologous proteingenus -4with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >98%, andthe above-mentionedmutant amino acidposition is same as E1-2(306-605). Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1proteinBovineYP_009272574.1Compare thepapillomavirushomologous protein-13with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >98%.Compare theabove-mentionedmutant amino acid withE1-2 (306-605), asparticacid (D) was replacedby glutamate (E) atamino acid 530 as ahomologousreplacement. Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1BovineAIN81126.1Compare theregulatorypapillomavirushomologous proteinprotein-13with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >98%.Compare theabove-mentionedmutant amino acid withE1-2 (306-605), asparticacid (D) was replacedby glutamate (E) atamino acid 530 as ahomologousreplacement. Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1 proteinBovineAGR88554.1Compare thepapillomavirushomologous protein-2with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >97%, andthe above-mentionedmutant amino acidposition is same as E1-2(306-605). Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1BovineAMZ04139.1Compare thepapillomavirushomologous protein-2with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >97%, andthe above-mentionedmutant amino acidposition is same as E1-2(306-605). Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1 proteinBovineAFQ90258.1Compare thepapillomavirushomologous protein-1with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >96%.Compare theabove-mentionedmutant amino acidposition with E1-2(306-605), aspartic acid(D) was replaced byglutamate (E) at aminoacid 530 as ahomologousreplacement. Therefore,the protein could alsoadopt the followingmutation scheme toimprove the poreperformance.E1BovineAKB94040.1Compare thepapillomavirushomologous protein-14with the bovinepapillomavirusdouble-stranded DNAhelicase E1 described inthe invention (NCBIaccession numberP03116), the sequencesimilarity is >75%.Compare theabove-mentionedmutant amino acidposition with E1-2(306-605), aspartic acid(D) was replaced byglutamate (E) at aminoacid 530 as ahomologousreplacement; lysine (K)was replaced byarginine (R) at aminoacid 417 as ahomologousreplacement; histidine(H) was replaced byasparagine (N) at aminoacid 323 as anon-homologousreplacement, Thereplacement fromhydrophobic aminoacids to hydrophilicones is useless;Glutamate (E) wasreplaced by glycine (G)at amino acid 392,replacing acidic aminoacids with neutral aminoacids, which is notbeneficially replaced;lysine (K) was replacedby leucine (L) at aminoacid 396, replacingbasic amino acids withneutral amino acids,which is not beneficiallyreplaced; lysine (K) wasreplaced by Glutamate(E) at amino acid 387,replacing basic aminoacids with acidicamino acid, which is notbeneficially replaced;Therefore, the proteincould also adopt thefollowing mutationscheme to improve thepore performance.

From the crystal structure and functional domain of the protein, we know that the conservative region of the protein is distributed in amino acids 1-121 and amino acids 166-575. In the two conservative regions, amino acids 407-557 are SF3 helicase functional domains, 413-457 amino acids belong to the AAA+ family of ATP hydrolases, and 142-308 amino acids are DNA binding domains. The amino acids 84-86 and 105-108 are respectively nucleic acid positioning signal regions; we identified some of the homologous proteins of these proteins, which have similar structures and same function (unwinding double-stranded DNA under the action of ATP hydrolysis energy). It is expected that these homologous proteins will also form stable pores on the artificial lipid membrane after adopting the described mutation and have the same on-membrane nucleic acid unwinding function as E1-1 (306-577), becoming a tool for the sensing of nucleic acid and small molecules.

According to the crystal structure analysis, the main helical functional domain of bovine papillomavirus double-stranded DNA helicase protein exists in the 308-577 amino acid segment. The invention constructs and purifies E1's truncated polypeptide E1-1 (306-577) in vitro. Using short-chained double-stranded DNA (dsDNA), the activity of ATP-dependent helicase in its hexamer is verified under specific conditions in vitro.

The invention further utilizes the truncated polypeptide E1-1 (306-577) to prepare nanopores. As mentioned in this invention, the hexameric pore completes the translocation of ssDNA, and the probability of translocation of single-stranded DNA (ssDNA) is slightly different.

The invention obtains another truncated body E1-2 (306-605) of E1 in further research. The protein can successfully form a stable nanopores on artificial lipid membrane under various conductive buffer conditions in vitro.

The invention also studies the following nucleic acid sensing using patch-clamp technology. Structural analysis shows that the inner diameter of the nanopore formed by E1-2 protein is about 1.3 nm, and the length of membrane region and the diameter of opening of the protein are about 3.2 nm and 15 nm respectively.

The experiment shown that under the buffer conditions of 1M, 0.3 mM KCl, and PBS, E1 (306-605) can self-assemble into a hexamer on an artificial lipid membrane and firmly form a pore on the membrane. After applying the transmembrane voltage on both sides of the membrane, generates a current passing through the pore. In the subsequent ssDNA transport experiment, it is found that short-chained ssDNA can trigger a series of current blocking phenomena, but using dsDNA to repeat the same experiment does not cause similar phenomena. The transmembrane current is stable, and these phenomena are consistent with the diameter of E1. The diameter of dsDNA is about 2 nm, while the inner diameter limit of E1 is about 1.3 nm, so when the dsDNA with flat end is driven by the transmembrane voltage, it is captured by E1. In most cases, either a continuously blocking with a blockage of more than 50% of the pore or the immediate opening of the pore after blocking occurs; When dsDNA with a single arm is captured by E1 driven by transmembrane voltage in PBS buffer or buffer with equal salt concentration as PBS, a specific concentration of ATP and Mg2+are added. Combined with E1's own helicase activity and its characteristics as a biological nanopore, it is shown that the double chain unwinding on the artificial lipid membrane was completed in vitro using the pore formed by E1-2 (306-605).

The mutants of E1-1 (306-577) and E1-2 (306-605) also have the same or similar functions and are also within the scope of protection of the invention.

It was found in the study that the mutants of E1-1 (306-577) and E1-2 (306-605) exhibited improved properties for subsequent nanopore incorporation and capture of negatively charged nucleic acids and ssDNA translocation. Specifically, the mutants show surprisingly easier artificial liposome insertion characteristics, stable presence on lipid membranes, and non-specific current fluctuations are further reduced due to the improvement of signal-to-noise ratio of open current caused by the pore. The capture efficiency of ssDNA at the C end of the pore was further improved. In the process of nucleic acid translocation, the inherent force of the pore cavity on ssDNA is weakened, and the translocation current presents a more rapid and regular peak pattern. Another advantage of the mutant is that the mutation does not affect the characteristics of the pore itself as a DNA helicase.

Therefore, the invention provides a mutated E1-1 (306-577) monomer or a mutated E1-2 (306-605) monomer, which contains variants of the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.3. Wherein the variant contains at least one of the following mutations (the number of bits of each of the following mutation sites is calculated from the first position of the total length of bovine papilloma virus double-stranded DNA helicase. The total length amino acid sequence of bovine papilloma virus double-stranded DNA helicase is shown in SEQID No. 5):

(a) The amino acid at 479 position is histidine (H), lysine (K), serine (S), asparagine (N), threonine (T);

(b) The amino acid at 489 position is histidine (H), lysine (K), asparagine (N), threonine (T) and serine (S);

(c) The amino acid at 530 position is histidine (H), lysine (K), serine (S), asparagine (N), threonine (T);

(d) The amino acid at 529 position is glutamine (Q) and lysine (K);

(e) The amino acid at 525 position is histidine (H), lysine (K), serine (S), leucine (L), threonine (T);

(f) The amino acid at 504 position is asparagine (N), lysine (K), arginine (R), serine (S), threonine (T), phenylalanine (f), tyrosine (Y), tryptophan (W);

(g) The amino acid at 328 position is glutamine (Q), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W);

(h) The amino acid at 360 position is asparagine (N), lysine (K), arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W);

(i) The amino acid at 322 position is asparagine (N), leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), glutamine (Q);

(j) The amino acid at 372 position is glutamine (Q), leucine (L), isoleucine (I), valine (V), proline (P), phenylalanine (F), tryptophan (W), asparagine (N);

(k) The amino acid at 342 position is asparagine (N), leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), glutamine (Q);

(l) The amino acid at 334 position is asparagine (N), leucine (l), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), glutamine (Q);

(m) The amino acid at 392 position is asparagine (N), leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), glutamine (Q);

(n) The amino acid at 408 position is asparagine (N), leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), glutamine (Q);

(o) The amino acid at 396 position is leucine (L), isoleucine (I), valine (V), phenylalanine (F) and tryptophan (W). Alanine (A) and glycine (G);

(p) The amino acid at 570 position is valine (V), phenylalanine (F), tryptophan (W), alanine (A), glycine (G), leucine (L), isoleucine (I);

(q) The amino acid at 574 position is tryptophan (W), alanine (A), glycine (G), leucine (L), isoleucine (I), valine (V) and phenylalanine (F);

(r) The amino acid at 417 position is leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), alanine (A) and glycine (G);

(s) The amino acid at 421 position is Valine (V), phenylalanine (F), tryptophan (W), alanine (A), glycine (G), leucine (L) and isoleucine (I);

(t) The amino acid at 383 position is valine (V), phenylalanine (F), tryptophan (W), alanine (A), glycine (G), leucine (L), isoleucine (I);

(u) The amino acid at 387 position is histidine (H), phenylalanine (F), tryptophan (W);

(v) The amino acid at 323 position is leucine (L), isoleucine (I), valine (v), phenylalanine (F), tryptophan (W), alanine (A), glycine (G).

(w) The amino acid at 324 position is leucine (L), isoleucine (I), proline (P) and valine (V);

(x) The amino acid at 565 position is threonine (T), serine (S) and tyrosine (Y);

(y) The amino acid at 426 position is leucine (L), isoleucine (I), valine (V), phenylalanine (F), tryptophan (W), alanine (A), glycine (G).

The invention provides a mutated E1-1 (306-577) monomer or a mutated E1-2 (306-605) monomer. The above-mentioned E1-1 (306-577) variants or E1-2 (306-605) variant monomers can be used to form the conductive membrane pore in this invention. Mutated E1-1 (306-577) monomer or E1-2 (306-605) monomer is a monomer whose sequence is different from that of wild E1-1 (306-577) monomer or E1-2 (306-605) monomer and retains the ability of forming pore. The methods used to confirm the capability of mutant monomers to form pores are consistent with methods mentioned in this invention.

The mutant E1-1 (306-577) monomer or E1-2 (306-605) monomer has multiple advantages over a wild type monomer in forming a pore on artificial lipid membrane. Specifically, the pore constructed by mutated monomer is more likely to capture nucleic acids and other negatively charged small molecules than wild type, in addition it shows an increase in current range, making the inner cavity of the pore narrower than that of wild type, so that the current detection in the pore is more extensive and sensitive.

In addition, when the mutated E1-1 (306-577) or E1-2 (306-605) monomers form a pore on an artificial lipid membrane, the transmembrane region of the pore is more likely to stably exist on the membrane and the signal-to-noise ratio of open current is higher than that of wild type. Surprisingly, when nucleic acid moves through some pores constructed by mutated E1-1 (306-577) or E1-2 (306-605) monomers, the current decreases even more. This makes it possible to use this pore to identify the relationship between nucleic acid sequence and the current descent platform when the nucleic acid moves through the pore, and further makes this mutant pore have the potential to apply to nucleic acid sequencing in the future. The improvements of nucleic acid reading properties of the mutants are realized through three main mechanisms, namely through changing:1. Space (the increase or decrease of mutated amino acid residues);2. Charge (for example, induces +ev charge to enhance the interaction between nucleic acids and amino acids);3. Non-covalent bonding (for example, induces amino acids that can bind with certain nucleotides with hydrogen bonds);

Either or more of these three mechanisms may be the reason why the pore of the invention has nucleotide reading property in the future.

The site mutation of the protein in the invention is mainly based on the following reasons:

In wild type E1-1 (306-577) or E1-2 (306-605), the N terminal is open at the narrower end, the C terminal is open at the wider end, and the hexamer forms a standard mushroom structure. A large number of alkaline amino acids are distributed in the inner cavity of the forming pore, which binds with negative phosphate group of ssDNA. Therefore, When the wild type E1-1 (306-577) or E1-2 (306-605) monomer is inserted into the artificial lipid membrane to form an conducting pore through which ions can freely pass it was found that the pore would fall from the lipid membrane at a long period of applied transmembrane voltage. The recorded open current baseline fluctuated, and small non-specific peak current is appeared occasionally when no molecule to be measured passes through the pore.

We use mutated E1-1 (306-577) or E1-2 (306-605) monomer to improve the above defects. For example, some acidic amino acids at the wider C terminal like aspartic acid (D) at 479 and 489 are mutated to histidine (H) or lysine (K) to increase the capture efficiency of negatively charged nucleic acid at the C terminal of the pore. When mutated to asparagine (N), the density of negatively charged amino acids at C-terminal opening decreased, and the electrostatic antagonism to nucleic acids (mainly) or negatively charged substances to be measured is correspondingly reduced, which indirectly increases the capture rate of the pore.

Another example is that we mutate some acidic amino acids in the pore cavity such as aspartic acid (D) at 504, glutamate (E) at 328, to asparagine (N), lysine (K), arginine (R), Glutamine (Q), etc. Mutating acidic amino acids (D, E) to neutral amino acids, on the one hand, can significantly reduce the electrostatic antagonism during nucleic acid translocation, on the other hand, When the neutral amino acid is phenylalanine (F), tryptophan (W), or tyrosine (Y) with a large steric hindrance, due to the larger spatial position within the formed cavity, the diameter of the non-restricted site in the cavity is significantly reduced. At the same time, when the density of benzene ring inside the substituted cavity increases, π electrons accumulate to make amino acids align more closely and the internal contraction of the cavity is obvious. Of course, these two reductions are double-edged swords in detection. When the neutral amino acid is a hydroxyl group, such as serine (S) or threonine (T), because hydroxyl oxygen can bond with nucleotides by forming hydrogen, the nucleic acid is able to enter the cavity from the C terminal to translocate more slowly. This mechanism will have more beneficial value in the subsequent development of nucleic acid sequencing.

Mutating aspartic acid (D) at 322, 342, 334, glutamate (E) at 372, lysine (K) at 396, 383, histidine (H) at 323 and arginine (R) at 574 in the transmembrane region to neutral amino acid Leucine (L), Glycine (G), alanine (A), etc. enable transmembrane region of the pore more easily and stably embed in the hydrophobic layer of lipid bilayer composed of long fatty acid chains, thus greatly weaken the open current baseline fluctuations and low frequency noise caused by instability of pore inserting. The specific mutation site is shown in table 3 below.

TABLE 3Mutant siteslocationsubstitutionadvantageD479HThe antagonism of DNA decreasesD479KThe capture of DNA improvesD479SPore is stableD479NThe antagonism of DNA decreasesD479TPore is stableD489HThe antagonism of DNA decreasesD489KThe capture of DNA improvesD489NThe antagonism of DNA decreasesD489TPore is stableD489SPore is stableD530HThe antagonism of DNA decreasesD530KThe capture of DNA improvesD530NThe antagonism of DNA decreasesD530TPore is stableD530SPore is stableD529HPore is stableD529KThe capture of DNA improvesD525HPore is stableD525KThe capture of DNA improvesD525NThe antagonism of DNA decreasesD525TPore is stableD525SPore is stableD504NDNA transports easily, the signal to noise ratioimprovesD504KDNA transports easilyD504RDNA transports easilyD504YHydrogen bonding, more compact cavityD504SHydrogen bondingD504THydrogen bondingE328HDNA transports easily, the signal to noise ratioimprovesE328KDNA transports easilyE328RDNA transports easilyE328SHydrogen bondingE328THydrogen bondingE328WMore compact cavityE328FMore compact cavityE328YMore compact cavity, hydrogen bonding,detection range and sensitivity increaseD360NDNA transports easily, the signal to noise ratioimprovesD360KDNA transports easilyD360RDNA transports easilyD360SHydrogen bondingD360THydrogen bondingD360WMore compact cavityD360FMore compact cavityD360YMore compact cavity, hydrogen bonding,detection range and sensitivity increaseD322NPore is stable, the signal to noise ratio improvesD322LPore is stable, the signal to noise ratio improvesD322IPore is stable, the signal to noise ratio improvesD322VPore is stableD322FPore is stableD322WPore is stable, the signal to noise ratio improvesD322HPore is stable, the signal to noise ratio improvesE372HPore is stableE372LPore is stable, the signal to noise ratio improvesE372IPore is stable, the signal to noise ratio improvesE372VPore is stableE372PPore is stableE372FPore is stable, the signal to noise ratio improvesE372WPore is stable, the signal to noise ratio improvesE372NPore is stableD342NPore is stableD342LPore is stable, the signal to noise ratio improvesD342IPore is stableD342VPore is stableD342FPore is stable, the signal to noise ratio improvesD342WPore is stable, the signal to noise ratio improves,the opening current level is stableD342HPore is stableD334NPore is stableD334LPore is stable, the signal to noise ratio improvesD334IPore is stableD334VPore is stableD334FPore is stable, the signal to noise ratio improves,the opening current level is stableD334WPore is stable, the signal to noise ratio improves,the opening current level is stableD334HPore is stableD392NPore is stableD392LPore is stable, the signal to noise ratio improvesD392IPore is stableD392VPore is stableD392FPore is stableD392WPore is stable, the opening current level is stableD392HPore is stableD408NPore is stableD408LPore is stable, the signal to noise ratio improvesD408IPore is stableD408VPore is stableD408FPore is stable, the signal to noise ratio improvesD408WPore is stable, the signal to noise ratio improvesD408HPore is stableK396LPore is stableK396IPore is stableK396VPore is stableK396FPore is stable, the signal to noise ratio improvesK396WPore is stable, the signal to noise ratio improves,the opening current level is stableK396APore is stableK396GPore is stableR570VPore is stableR570FPore is stable, the signal to noise ratio improves,the opening current level is stableR570WPore is stable, the signal to noise ratio improvesR570APore is stableR570GPore is stableR570LPore is stable, the signal to noise ratio improvesR570IPore is stableR574FPore is stable, the signal to noise ratio improves,the opening current level is stableR574WPore is stable, the signal to noise ratio improvesR574APore is stableR574GPore is stableR574LPore is stable, the signal to noise ratio improvesR574IPore is stableR574VPore is stableK417LPore is stable, the signal to noise ratio improvesK417IPore is stableK417VPore is stableK417FPore is stable, the signal to noise ratio improvesK417WPore is stable, the signal to noise ratio improves,the opening current level is stableK417APore is stableK417GPore is stableK421VPore is stableK421FPore is stable, the signal to noise ratio improves,the opening current level is stableK421WPore is stable, the signal to noise ratio improves,the opening current level is stableK421APore is stableK421GPore is stableK421LPore is stable, the signal to noise ratio improvesK421IPore is stable, the signal to noise ratio improvesK383VPore is stableK383FPore is stable, the signal to noise ratio improvesK383APore is stableK383GPore is stableK383LPore is stable, the signal to noise ratio improvesK383IPore is stable, the signal to noise ratio improvesK383WPore is stable, the signal to noise ratio improves,the opening current level is stableK387HPore is stable, the signal to noise ratio improvesK387FPore is stable, the signal to noise ratio improves,the opening current level is stableK387WPore is stable, the signal to noise ratio improves,the opening current level is stableH323LPore is stable, the signal to noise ratio improvesH323IPore is stable, the signal to noise ratio improvesH323VPore is stableH323FPore is stable, the signal to noise ratio improves,the opening current level is stableH323WPore is stable, the signal to noise ratio improves,the opening current level is stableH323APore is stableH323GPore is stableK324LPore is stable, the signal to noise ratio improvesK324IPore is stable, the signal to noise ratio improvesK324PPore is stable, the signal to noise ratio improves,the opening current level is stableK324VPore is stableK565TPore is stableK565SPore is stableK565YPore is stableK426LPore is stable, the signal to noise ratio improvesK426IPore is stable, the signal to noise ratio improvesK426VPore is stableK426FPore is stable, the signal to noise ratio improves,the opening current level is stableK426WPore is stable, the signal to noise ratio improves,the opening current level is stableK426APore is stableK426GPore is stable

The present invention is further elaborated by embodiments as follows.

Embodiment 1 Construction and Expression Purification of E1 Protein and its Mutant Recombinant Vector

The amino acid sequence (SEQ ID No. 3) of truncated E1-1 (306-577):LQTEKFDFGTMVQWAYDHKYAEESKIAYEYALAAGSDSNARAFLATNSQAKHVKDCATMVRHYLRAETQALSMPAYIKARCKLATGEGSWKSILTFFNYQNIELITFINALKLWLKGIPKKNCLAFIGPPNTGKSMLCNSLIHFLGGSVLSFANHKSHFWLASLADTRAALVDDATHACWRYFDTYLRNALDGYPVSIDRKHKAAVQIKAPPLLVTSNIDVQAEDRYLYLHSRVQTFRFEQPCTDESGEQPFNITDADWKSFFVRLWGRLDL.The coding gene sequence (SEQ ID No. 4) of the truncated E1-1 (306-577):Ttgcagaccgagaaattcgacttcggaactatggtgcaatgggcctatgatcacaaatatgctgaggagtctaaaatagcctatgaatatgctttggctgcaggatctgatagcaatgcacgggcttttttagcaactaacagccaagctaagcatgtgaaggactgtgcaactatggtaagacactatctaagagctgaaacacaagcattaagcatgcctgcatatattaaagctaggtgcaagctggcaactggggaaggaagctggaagtctatcctaactttttttaactatcagaatattgaattaattacctttattaatgattaaagctctggctaaaaggaattccaaaaaaaaactgtttagcatttattggccctccaaacacaggcaagtctatgctctgcaactcattaattcatttttgggtggtagtgttttatcttttgccaaccataaaagtcacttttggcttgcttccctagcagatactagagctgctttagtagatgatgctactcatgcttgctggaggtactttgacacatacctcagaaatgcattggatggctaccctgtcagtattgatagaaaacacaaagcagcggttcaaattaaagctccacccctcctggtaaccagtaatattgatgtgcaggcagaggacagatatttgtacttgcatagtcgggtgcaaacctttcgctttgagcagccatgcacagatgaatcgggtgagcaaccttttaatattactgatgcagattggaaatctttttttgtaaggttatgggggcgtttagacctg.Vector plasmid: pGEX-6p-1.Double digestion: BamH I, Xho I.Primer (lowercase letters are restriction sites):Forward primer: SEQ ID No. 10;CGggatccTTGCAGACCGAGAAAT.Reverse primer: SEQ ID No. 11CCGctcgagTTAATGATGATGGTGATGATGCAGGTCTAAACGCCCC.The amino acid sequence (SEQ ID No. 1) of truncated E1-2 (306-605):LQTEKFDFGTMVQWAYDHKYAEESKIAYEYALAAGSDSNARAFLATNSQAKHVKDCAMVRHYLRAETQALSMPAYIKARCKLATGEGSWKSILTFFNYQNIELITFINALKLWLKGIPKKNCLAFIGPPNTGKSMLCNSLIHFLGGSVLSFANHKSHFWLASLADTRAALVDDATHACWRYFDTYLRNALDGYPVSIDRKHKAAVQIKAPPLLVTSNIDVQAEDRYLYLHSRVQTFRFEQPCTDESGEQPFNITDADWKSFFVRLWGRLDLIDEEEDSEEDGDSMRTFTCSARNTNAVD.The coding gene sequence (SEQ ID No. 2) of the truncated E1-2 (306-605):ttgcagaccgagaaattcgacttcggaactatggtgcaatgggcctatgatcacaaatatgctgaagagtctaaaatagcctatgaatatgctttggctgcaggatctgatagcaatgcacgggcttttttagcaactaacagccaagctaagcatgtgaaggactgtgcaactatggtaagacactatctaagagctgaaacacaagcattaagcatgcctgcatatattaaagctaggtgcaagctggcaactggggaaggaagctggaagtctattctaactttttttaattatcagaatattgaattaattacctttattaatgctttaaagctctggctaaaaggaattccaaaaaaaaactgtttagcatttattggccctccaaacacaggcaagtctatgctctgcaactcattaattcattttttgggtggtagtgttttatcttttgccaaccataaaagtcacttttggcttgcttccctagcagatactagagctgctttagtagatgatgctactcatgcttgctggaggtactttgacacatacctcagaaatgcattggatggctaccctgtcagtattgatagaaaacacaaagcagcggttcaaattaaagctccacccctcctggtaaccagtaatattgatgtgcaggcagaggacagatatttgtacttgcatagtcgggtgcaaacctttcgctttgagcagccatgcacagatgaatcgggtgagcaaccttttaatattactgatgcagattggaaatctttttttgtaaggttatgggggcgtttagacctgattgacgaggaggaggatagtgaagaggatggagacagcatgcgaacgtttacatgtagcgcaagaaacacaaatgcagttgattga.Vector plasmid: pGEX-6p-1.Double digestion: BamH I, Xho I.Primer (lowercase letters are restriction sites):Forward primer 1: SEQ ID No. 12AGTTCTGTTCCAGGGGCCCCTGggatccTTGCAGACCGAGAAATTCGACTTCG.Reverse primer 1: SEQ ID No. 13TCAGTCAGTCACGATGCGGCCGctcgagTTAATCAACTGCATTTGTGTTTCTTGCGCTACATGTAAACGTTCGCA.The amino acid sequence of E1-2 mutant 1 (K421L) is shown in SEQ ID No. 6:LQTEKFDFGTMVQWAYDHKYAEESKIAYEYALAAGSDSNARAFLATNSQAKHVKDCATMVRHYLRAETQALSMPAYIKARCKLATGEGSWKSILTFFNYQNIELITFINALKLWLLGIPKKNCLAFIGPPNTGKSMLCNSLIHFLGGSVLSFANHKSHFWLASLADTRAALVDDATHACWRYFDTYLRNALDGYPVSIDRKHKAAVQIKAPPLLVTSNIDVQAEDRYLYLHSRVQTFRFEQPCTDESGEQPFNITDADWKSFFVRLWGRLDLIDEEEDSEEDGDSMRTFTCSARNTNAVD.The nucleotide sequence of E1-2 mutant 1 is shown in SEQ ID No. 8:GGATCCTTGCAGACCGAGAAATTCGACTTCGGAACTATGGTGCAATGGGCCTATGATCACAAATATGCTGAGGAGTCTAAAATAGCCTATGAATATGCTTTGGCTGCAGGATCTGATAGCAATGCACGGGCTTTTTTAGCAACTAACAGCCAAGCTAAGCATGTGAAGGACTGTGCAACTATGGTAAGACACTATCTAAGAGCTGAAACACAAGCATTAAGCATGCCTGCATATATTAAAGCTAGGTGCAAGCTGGCAACTGGGGAAGGAAGCTGGAAGTCTATCCTAACTTTTTTTAACTATCAGAATATTGAATTAATTACCTTTATTAATGCTTTAAAGCTCTGGCTACTGGGAATTCCAAAAAAAAACTGTTTAGCATTTATTGGCCCTCCAAACACAGGCAAGTCTATGCTCTGCAACTCATTAATTCATTTTTTGGGTGGTAGTGTTTTATCTTTTGCCAACCATAAAAGTCACTTTTGGCTTGCTTCCCTAGCAGATACTAGAGCTGCTTTAGTAGATGATGCTACTCATGCTTGCTGGAGGTACTTTGACACATACCTCAGAAATGCATTGGATGGCTACCCTGTCAGTATTGATAGAAAACACAAAGCAGCGGTTCAAATTAAAGCTCCACCCCTCCTGGTAACCAGTAATATTGATGTGCAGGCAGAGGACAGATATTTGTACTTGCATAGTCGGGTGCAAACCTTTCGCTTTGAGCAGCCATGCACAGATGAATCGGGTGAGCAACCTTTTAATATTACTGATGCAGATTGGAAATCTTTTTTTGTAAGGTTATGGGGGCGTTTAGACCTGATTGACGAGGAGGAGGATAGTGAAGAGGATGGAGACAGCATGCGAACGTTTACATGTAGCGCAAGAAACACAAATGCAGTTGATTAACTCGAG.The amino acid sequence of E1-2 mutant 2 (H323W) is shown in SEQ ID No. 7:LQTEKFDFGTMVQWAYDWKYAEESKIAYEYALAAGSDSNARAFLATNSQAKHVKDCATMVRHYLRAETQALSMPAYIKARCKLATGEGSWKSILTFFNYQNIELITFINALKLWLLGIPKKNCLAFIGPPNTGKSMLCNSLIHFLGGSVLSFANHKSHFWLASLADTRAALVDDATHACWRYFDTYLRNALDGYPVSIDRKHKAAVQIKAPPLLVTSNIDVQAEDRYLYLHSRVQTFRFEQPCTDESGEQPFNITDADWKSFFVRLWGRLDLIDEEEDSEEDGDSMRTFTCSARNTNAVD.The nucleotide sequence of E1-2 mutant 2 is shown in SEQ ID No. 9:GGATCCTTGCAGACCGAGAAATTCGACTTCGGAACTATGGTGCAATGGGCCTATGATTGGAAATATGCTGAGGAGTCTAAAATAGCCTATGAATATGCTTTGGCTGCAGGATCTGATAGCAATGCACGGGCTTTTTTAGCAACTAACAGCCAAGCTAAGCATGTGAAGGACTGTGCAACTATGGTAAGACACTATCTAAGAGCTGAAACACAAGCATTAAGCATGCCTGCATATATTAAAGCTAGGTGCAAGCTGGCAACTGGGGAAGGAAGCTGGAAGTCTATCCTAACTTTTTTTAACTATCAGAATATTGAATTAATTACCTTTATTAATGCTTTAAAGCTCTGGCTAGGAATTCCAAAAAAAAACTGTTTAGCATTTATTGGCCCTCCAAACACAGGCAAGTCTATGCTCTGCAACTCATTAATTCATTTTTTGGGTGGTAGTGTTTTATCTTTTGCCAACCATAAAAGTCACTTTTGGCTTGCTTCCCTAGCAGATACTAGAGCTGCTTTAGTAGATGATGCTACTCATGCTTGCTGGAGGTACTTTGACACATACCTCAGAAATGCATTGGATGGCTACCCTGTCAGTATTGATAGAAAACACAAAGCAGCGGTTCAAATTAAAGCTCCACCCCTCCTGGTAACCAGTAATATTGATGTGCAGGCAGAGGACAGATATTTGTACTTGCATAGTCGGGTGCAAACCTTTCGCTTTGAGCAGCCATGCACAGATGAATCGGGTGAGCAACCTTTTAATATTACTGATGCAGATTGGAAATCTTTTTTTGTAAGGTTATGGGGGCGTTTAGACCTGATTGACGAGGAGGAGGATAGTGAAGAGGATGGAGACAGCATGCGAACGTTTACATGTAGCGCAAGAAACACAAATGCAGTTGATTAACTCGAG.A mutant recombinant plasmid is constructed, and the mutated vector insertsinto the vector plasmid pGEX-6P-1 by PCR cloning, and the double restriction sitesare 5′BamhI and 3′XhoI respectively.Forward primer 2: SEQ ID No. 14AGTTCTGTTCCAGGGGCCCCTGggatccTTGCAGACCGAGAAATTCGACTTCG.Reverse primer 2: SEQ ID No. 15TCAGTCAGTCACGATGCGGCCGctcgagTTAATCAACTGCATTTGTGTTTCTTGCGCTACATGTAAACGTTCGCA.

The entire bovine papillomavirus genome is amplified using the above PCR primers (as described above), and then the vector pGEX-6p-1 and the resulting PCR product are digested with two restriction enzymes BamH I and Xho I. The vector carried the GST-tag affinity containing two restriction sites of BamH I and Xho I respectively. Finally, the double-digested target gene fragment and the plasmid vector are religated into a circular recombinant plasmid by T4 ligase.

The detection of recombinant plasmid is to preliminarily screen whether the recombinant plasmid is successfully transformed or not by using ampicillin resistance of the recombinant vector after the transformed plasmid intoE. coliDH5a. After colony PCR, the nucleic acid gel electrophoresis is used to preliminarily verify whether the target fragment is ligated into the vector (as shown inFIG.1, pGEX-6p-1 is 4984 bp in length, and the E1-1 gene sequence is 816 bp in length, so two bands are obtained after digestion, respectively 4990 bp and 822 bp). Then sequencing results obtained by the gene sequencing are used to confirm whether the recombination is completed and no mutation occurs.

The specific expression and purification of E1 protein: recombinant plasmid pGEX-6p-1-N-GST is transformed intoE. coliexpression strain BL21 for protein expression. The aboveE. coliis cultured overnight in 11 mL LB medium containing 50 μg/mL ampicillin at 37° C. and 220 rpm. Then, 10 mL of culture solution is injected into 1 L of fresh LB medium containing 50 μg/ml ampicillin, and 0.5 mM (final concentration) IPTG (Isopropyl β-D-Thiogalactoside) is added to induct when the bacterial concentration reaches 0.6-0.8 units (OD600). After induction for 16 hours at 16° C., the bacteria are harvested by centrifugation at 4000 rpm for 20 minutes, and the suspension is resuspended in protein buffer 20 mM tris-HCl (pH 7.3), 200 mM NaCl. After the bacteria are broken by high pressure four times, the supernatant is harvested by centrifugation at 16000 rpm for 40 minutes. Then the supernatant is filtered through a 0.45 μm pore size filter and the target protein is purified by GST affinity chromatography. After the hybrid protein is eluted, the target protein is digested on the PSP enzyme column for 2 hours, and then the target protein is elute with protein buffer.

The purity of the target protein is high after purification by GST affinity chromatography. Finally, the molecular sieve is used to separate the polymer and the monomer, and the first peak protein is the pure E1. Simultaneously, from the analysis of the peak position of molecular sieve, the obtained E1 is in the form of hexamer, and the wild E1 form is very similar. The remaining protein samples are tested by polyacrylamide gel electrophoresis to meet the expected size.FIGS.2and3are the polyacrylamide gel electrophoresis patterns of the molecular sieve peaks of E1-1 and E1-2 57 respectively. The corresponding monomer molecular weights are 57 KD and 59 KD respectively. The second tube (E1-1) and the sixth tube (E1-2) are used in the subsequent electrophysiological experiments.

The methods for gene cloning and expression purification of two mutants refers to wild type E1 (306-605), and the first appeared peak-tube after molecular sieve collection is the mutant E1. The remaining protein samples are tested by polyacrylamide gel electrophoresis compared with WT-E1 (306-605), since only one amino acid is mutated, the molecular weight of the mutant is very close to the WT-E1 (306-605) protein. Moreover, they are almost in a same position on the gel electrophoresis strip, as shown inFIG.21.

Embodiment Two, Fluorescent Labeling and Membrane Fusion Experiments of Protein Truncations and their Mutants

Fluorescently labeled truncated E1-2 (306-605) protein: The material used is a fluorine-labeled fluorescein isothiocyanate (FITC) conjugate kit (Sigma, St. Louis, Missouri). According to the kit description, the excess FITC is removed by column chromatography, and the FITC-linked E1 is eluted with protein buffer (20 mM tris-HCl (pH7.3), 200 mM NaCl). Verified FITC labels E1 (306-605) with SDS-PAGE (Figure omitted, the position of FITC labeled E1-2 on SDS-PAGE is slightly higher than that of unlabeled E1-2).

Preparation of vesicles (SUVs) containing truncated bodies E1-2 (306-605): In order to prepare vesicles of about 0.1 um in size containing E1 protein, 1 mg/mL of DOPC is add into a vial. The aqueous solution containing protein E1 (concentration about 0.5 mg/ml) is added after nitrogen blow dry the chloroform, then hydrates the lipid membrane to make suspension, and this suspension is further extruded by Mini squeezer (purchased from AVANTI Co. Ltd, America) through polycarbonate (PC) films (diameter of 100 nm) to produce protein-SUVs. The resulted SUVs can be stored at 4° C. for a short term and at −80° C. for a long term; Only the SUVs without labeled by fluorescence shows similar form to vesicles of the same size under optical microscope. (The resulted protein-SUVs by Mini squeezer have a good uniformity).

The procedure of preparing giant vesicles containing truncated E1-2 (306-605): meanwhile, giant vesicles labeled with fluorescence could also be prepared. Mixing 1 ml of DOPC (1 mg/ml) or 1 ml of DPHPC (1 mg/ml) and 1% of Texas Red (purchased from Thermofisher Co. Ltd, America) in a brown bottle; then drying chloroform by nitrogen, or volatilizing chloroform in rotary evaporator in this procedure; next, putting DOPC or DPHPC obtained from above step in vacuum drier overnight; In order to acquire hybrid giant unilamellar vesicles (GUVs) inserted E1 protein, adding 200-300 Mm of sucrose solution which containing E1 (concentration about 0.5 mg/ml) to hydrate the lipid molecular layer.

Those vesicles labeled with Texas Red can be observed in fluorescence microscope. To prepare fluorescence-labeled protein complexes embedded with vesicles, FITC-labeled E1-2 obtained from previous procedure can be used here. The result of observing GUVs in fluorescence microscope shows that the size of vesicles with larger diameter obtained by rotary evaporator is not as uniform as that of SUVs prepared by squeezer. Some vesicle protein complex with diameter over 50 μm are found in latter nanopore insertion with artificial membrane trail, and these vesicles easily ruptures bilayer lipid membrane (artificial lipid membrane is formed on the surface of 50 μm-diameter hole which made up of polyacetal resin, purchased from Warner Co. Ltd, America).

Conducting membrane fusion experiment on the mutant of truncated E1-2 with the same method, the result shows that, consistent with WT-E1 (306-605), this protein can also be steady inserted into the artificial lipid bilayer and PMOXA-PDMS-PMOXA triblock copolymer. The procedure of the experiment is similar to that of WT-E1 (306-605), when the GUVs inserted with mutant E1 is obtained, the protein embedding on the membrane is observed by Cryo-EM, and as shown inFIG.22andFIG.23, the porin can steady exist on the membrane.

The double-labeled vesicle-embedded protein complex (red as the Texas red labeled vesicles, and green as the FITC labeled E1-2 protein) is observed under fluorescence microscope, and it is found that some labeled E1-2 protein shows irregular positioning on vesicle membrane, part of the “transmembrane region” as expected is inserted into the phospholipid bilayer to form pores. This result also proves that in the conductance buffer, E1-2 can spontaneously aggregate on the artificial lipid membrane driven by voltage and can insert into the membrane in an expected direction to form a conductive pore.

Embodiment Three, the Electrophysiological Experiment of E1 Protein and its Mutant

Electrophysiological measurement: the instrument used in this embodiment is HEKA EPC-10 USB which integrates Amplifier and Digital-to-analog converter (DAC), and it has two electrodes named of pressure (voltage clamp) electrode and reference electrode, which are a pair of silver/silver chloride electrodes (Ag/AgCl). The instrument is used to measure the transmembrane current across both sides of the phospholipid bilayer. The sample frequency setup here is 2 kHz, and the frequency of low-pass filter is 1 kHz; the software Patch-master is used to collect the data, and Clampfit is utilized to analyze the collected data.

The fabrication of bilayer lipid membrane (BLM): The horizontal standard BLM fabricator and vertical standard BLM chamber (BCH-13A, purchased from Warner. Co. Ltd, America; Cups contain a 50 μm-diameter hole is custom made) are used to fabricate the BLM. A thin Teflon film with an aperture of 80-240 μm (TP-02 from Eastern Sci. LLC) and a Polyformaldehyde resin CUPS with an aperture of 50 μm are used as a partition to respectively separate BLM fabricator and BLM chamber into cis-compartment (working volume 250 μL) and trans-compartment (working volume 2.5 mL). After the aperture is pre-painted twice with 0.5 μL, 1 mg/ml of DOPC solution or DPHPC solution (the solvent is n-hexane) to ensure the complete coating of the entire edge of the aperture, the two compartments are filled with conducting buffers (5 mM Hepes/pH 7.5, with varying concentration of KCl solution, or PBS solution); Then, adding 25 mg/ml of DOPC solution or DPHPC solution (the solvent is n-hexane) near 50 μm aperture and it finds that the current falling from 10.2 nA to 0 pA.

The distribution of conductance of E1:

The vesicle-protein complex prepared previously forms a BLM embedded with E1-2 protein nanopore structure on the micropores of horizontal ptfe membrane (as described above) or vertical polyformaldehyde resin micropores.

The stock solution of vesicles/E1 complex prepared earlier is diluted by 10-20 times for the BLM experiments before use. For insertion of E1, around 2 μL of the diluted liposome solution is loaded into the cis-chamber; the other simple method is described below: a bubble containing some liposome at the pipette top is used to sweep the aperture gently in the cis-chamber, and the pore would form spontaneously. Conductance of E1 protein is measured in two methods: the first is under a constant transmembrane voltage (e.g. −70 mv), measuring current leap when multiple pores are embedded and dropped, and then counting data to calculate the distribution of conductance of pores. The second method is to apply a scanning voltage (e.g. −120 mv to 120 mv) to both sides of the membrane and calculate the pore conductance by calculating the rate of the current and voltage curve.

In this invention, large numbers of data are counted and the result demonstrates that, the single-pore conductance in 5 mM HEPES and 1 M KCl is around 1.34±0.07 nS (as shown inFIG.5); The single-pore current level and the two-pores current level are about 105 pA and 200 pA respectively, when given a +70 mV trans-voltage (as shown inFIG.4). A plot of the I-V curve is obtained under different ramping voltages and the single-pore conductance is measured around 1.5 nS (as shown inFIG.6). Under the condition of 5 mM HEPES and 0.5M KCl conductance buffer, the measured single-conductance of E1 is around 0.78 ns, at a transmembrane voltage of 50 mv, the current of a single E1 formed pore on the artificial lipid membrane is about 40 pA, and that of double pores is about 85 pA. Under I-V curve, the measured single-pore conductance is about 1.57/2 nS. In PBS as the conductance buffer, the conductance of the protein is 0.2±0.02 nS (as shown inFIG.7), and the current of single-pore is about 23 pA under a voltage of −70 mV.

The same trial on mutant E1 is conducted, and the result shows that under 0.5 M KCl, 10 Mm HEPES (pH 7.5), single-pore embedding causes about 75 pA of current ascending step when given a +100 mV bias voltage, as shown inFIG.24, after counting a large number of single-pore embedding and falling signals and under the scanning voltage of −200-200 mv, it is found that the single-pore conductance distribution is similar to that of WT-E1 (306-605) pores, both of which are about 1.3±0.07 nS. Moreover, at the scanning voltage, the scatter linear fitting results of current to voltage are good and the dispersion is low.

Embodiment Four, the Study on the Stability of the Pores of E1 Protein and its Mutant

The study on the stability of the pore of E1-2: in this invention, the stability of pores of E1-2 and the mutant of E1 on phospholipids membrane and high voltage-gating phenomenon are studied under positive and negative voltage respectively. The results depicted: no gating phenomenon is found for E1 pore under positive 300 mV and negative 300 mV, and E1 pore could exist on the phospholipids membrane steadily when given a high voltage. At the same time, it is found that when there are too much glycerol in the conductance buffer (used to preserve E1 protein), the membrane embedded protein complex become abnormally unstable, and it is extremely easy for the protein to fall off from the membrane or the membrane to rupture directly.

In the stability experiment of E1 protein mutant (K421L, H323W) at high voltage, the scanning voltage of −300-300 mv is adopted. It finds that at a higher voltage (±300), the pore remained stable on the membrane, and the conductance distribution does not change significantly, showing that the mutant E1 (K421L, H323W) also has stable conductance characteristics as WT-E1 (306-605).

Embodiment Five, Single-Stranded DNA/RNA Translocation Tests of E1 Protein and its Mutant

ssDNA used in this embodiment is 48 nt and purchased from Takara company, its detail sequence is depicted as below; The electrolyte is HEPES/1M KCl; In general, there are two methods to achieve nucleic acid transport under this condition.

Method 1: When E1 protein inserts into BLM, changing the voltage to 0 mV and adding the nucleic acids to be transported to the cis; Method 2: the nucleic acid to be transported is pre-mixed into the conductance buffer, and the cis and trans are mixed in the same amount. The translocation of DNA mainly depends on the applied transmembrane voltage. Without special instructions, method 1 is adopted for ssDNA translocation in this experiment, and the concentration of ssDNA in the translocation system is 100 nM/L.

In the case of the 48 nt ssDNA, the single-pore pore is embedded in the membrane, and the current blocking caused by translocation is about 65 picoamperes (transmembrane voltage is about 50 mv) (as shown inFIG.8). The current blocking ratio induced by translocation of nucleic acid in this segment is about 82% (Ib/I, Ib is the current blocking caused by nucleic acid translocation, unit is pA; I is the current change caused by the insertion of a single-pore protein into the phospholipid bilayer, unit is pA) The dwell time of ssDNA translocation is 2 ms (as shown inFIGS.29-30). In PBS, ssDNA translocation experiment shows similar results that under +100 mV transmembrane voltage, 24 nt ssDNA would induced around 80% blockage and 1-2 ms dwell time (as shown inFIGS.9-10).

Meanwhile, ssRNA translocation experiment is conducted like the ssDNA translocation experiment mentioned above. Noted that using DEPC solution and autoclave sterilization to prevent RNA degradation. The result shows that, similar to ssDNA translocation, ssRNA induced around 80% blockage, and under −50 mV the blocked current in PBS buffer system is about 8 pA, the dwell time is 1-2 ms.

As a comparison, the current trace in the system without nucleic acid but with only pores are static. One advantage of this pore is that the static current in the control group rarely interferes with the non-specific blocking. In the experiment, when nucleic acid premixed into the conductance buffer, amounts of translocation instantly found after the first insertion of the protein pore into the phospholipid membrane, and the blocking phenomenon becomes less and less. This result shows that when there is no stirring equipment in the sample tank, the occurrence of DNA translocation is delayed.

E1 protein mutant is translocated by ssDNA/ssRNA using the same conductance conditions and single stranded nucleic acid. Under the conductance condition of 1M KCl and 10 mM Hepes (pH7.5), applying the bias voltage of 120 mv, it finds that the blocking rate of ssDNA translocation is close to 78%, and the translocation time is close to 0.5 ms (as shown inFIGS.26and25), which approximates to the data of WT-E1 (306-605).

Embodiment Six, Blockage Experiment of dsDNA Inside E1

The double-stranded DNA (dsDNA) obtained from BamH1 enzyme is added to the cis-chamber, and the translocation buffer is 10 mM HEPES/1M KCl with ssDNA. The transfer signals are detected at 50 mv, 70 mv, 120 mv and 150 mv voltages respectively, and no dsDNA translocation is observed.

Embodiment Seven, Verification Experiment of E1 Nucleic Acid Translocation

Q-PCR is used to make the standard curve of nucleic acid amplification: dsDNA is prepared from annealing two ssDNA (80nt) and identified with 2% agarose gel, and extracted from gel. The concentration of dsDNA extracted from gel is determined by Qubit®3.0 (Thermo fisher scientific Co. Ltd, America) (This method is better than ultraviolet spectrophotometer in accuracy and precision); The resulting dsDNA of known concentration is diluted into 10 of 10-time concentration gradients, and then using Q-PCR (Premix SYBR-Takara Co. Ltd, Japan) to make the standard curve for the ct value of dsDNA concentration, so as to obtain the standard correlation (as shown inFIGS.11-12, Ct value has a good repeatability under each concentration gradient, and R2=0.99+. Therefore, this standard correlation can be used as a basis for the calculation of subsequent ssDNA translocation experiments.)

Quantitative analysis of ssDNA through pore by Q-PCR, as mentioned above, after E1 protein embedded into the membrane in cis-chamber, adding 50 nM 80 nt-DNA (the first method), or premixing 25 nM 48 nt-DNA into conductance buffers at both side chambers (the second method). As a negative control, 50 nM 80nt-DNA is added to cis-chamber without E1 protein, or the equivalent amount of 25 nM 80nt-DNA is added to both side chambers. Applying a voltage of −70 mV to both sides of the membrane and collecting two-chambers' conductance buffer for Q-PCR quantitative analysis after 10 min, 20 min, 40 min, 60 min and 90 min, respectively.

All nucleic acids involved in translocation experiments are described below: (purchased from Takara, Co. Ltd, Japan)

48 nt-DNA sequence is shown in SEQ ID No. 16:3′-TTT TTT TTT AAA AAA TTT TTT GGG GGG TTTTTT CCC CCC TTT TTT TTT-5′.Sequences of 44 nt-DNA and 44 nt-DNA 1 are shownin SEQ ID No. 17 and SEQ ID No. 18, respectively:(SEQ ID No. 17)5′-AGC TCC ACC CCT CCT GGT AAC CAG TTT TTT TTTTTT TTT TTT TT-3′.(SEQ ID No. 18)5′-TTT TTT TTT TTT TTT TTT TT CTG GTT ACC AGG AGGGGT GGA GCT-3′.24 nt-DNA sequence is shown in SEQ ID No. 19:5′-CTG GTT ACC AGG AGG GGT GGA GCT-3′.

Labeled nucleic acid chain and labeled nucleic acid chain 1 are described as SEQ ID No. 20 and SEQ ID No. 21:

(SEQ ID No. 20)5′-CCTACGCCACCAGCTCCGTAGG-3′ (5′-Cy5, 3′-BHQ2).(SEQ ID No. 21)5′-CCTACGGAGCTGGTGGCGTAGGTTTTTTTTTTTTTTTTTTTT-3′.

The sequences of dsDNA and dsDNA1 with dspacer (dspacer is a nucleotide having only a phosphate skeleton and no bases, therefor its spatial structure is smaller than normal nucleotides) are shown in SEQ ID No. 22 (three dspacer are inserted between the eleventh and twelfth bits) (the detail sequence is 5′-TTTTTTTTTTT XXXTTTTTTTTTTT-3′, and X represents dspacer) and SEQ ID No. 23:

(SEQ ID No. 22)5′-TTTTTTTTTTTTTTTTTTTTTT-3′.(SEQ ID No. 23)5′-AAAAAAAAAAAAAAAAAAAA-3′.

The sequence of nucleic acid used in quantitative translocation analysis by Q-PCR are shown in SEQ ID No. 24 and SEQ ID No. 25:

(SEQ ID No. 24)5′-CAG GCA GAG GAC AGA TAT TTG TAC TTG CAT AGTCGG GTG CAA ACC TTT CGC TTT GAG CAG CCA TGC ACAGAT GAA TCG GGT TTT TTT TTT TTT TTT TTTT.(SEQ ID No. 25)5′-CCC GAT TCA TCT GTG CAT GGC TGC TCA AAG CGAAAG GTT TGC ACC CGA CTA TGC AAG TAC AAA TAT CTGTCC TCT GCC TG.

The two primers (primer 1 and primer 2) used for quantitative translocation by Q-PCR are shown in SEQ ID no.26 and SEQ ID no.27, respectively:

(SEQ ID No. 26)5′-CAG GCA GAG GAC AGA TAT TT.(SEQ ID No. 27)5′-CCC GAT TCA TCT GTG CAT GG.

The annealing procedure of dsDNA is performed at PCR equipment, the annealing temperature is 95° C., and the annealing time is set up to 2 min; then the process of cooling is set up to gradient (almost 1° C./min), which lasts 1 h and 20 min. Lastly, the nucleic acid product is determined by agarose gel (as shown inFIG.13).

In this invention, dsDNA is acquired from circular plasmid Hag-C1 digested by BamH1, and it is purified by QIAGEN DNA purification kit the product 3800 bp dsDNA was conducted by QIAGEN DNA purification kit (QIAGEN, Co. Ltd, Germany).

Embodiment Eight, Unwinding Experiment of E1 In Vitro

The invention using E1 (306-577) to verify the helical activity of ds DNA of the protein hexamer in vitro.

The buffer solution in helicase experiment is 20 mM HEPES (pH 7.5), 0.7 mg/ml BSA, 5% Glycerol, 5 mM MgCl2, 3 mM DTT; And the reaction is initiated by adding 2 mM ATP; the substrate dsDNA in this trial is 22 bp labeled with 5′-fluor and 3′-quencher at the terminals of DNA which could form hairpin spontaneously.

In vitro, dsDNA ends flat cannot be directly unwind by this helicase due to the lack of other protein components contained in the virus itself to assist in DNA replication.

Synthesized dsDNA with a single stranded arm is used in the unwinding experiment in vitro and unwinding experiment on membrane mentioned later. E1 protein unwinding starts with the single-arm nucleic acid strand entering the pore. In the process of unwinding, excitation wavelength is 643 nm and emission wavelength is 667 nm, Multifunctional fluorescence microplate reader (Bio-Tek, Co. Ltd, America) is used to continuously monitor the fluorescence of the substrate (5 nM nucleic acid substrate) in the reaction system to visually observe the unwinding process. When dsDNA is unwound, one of the ssDNA terminal forms spontaneously hairpin structure, and fluorescence tag could be quenched by quencher tag.

The result demonstrates that, E1-2 has helicase activity in vitro, double-stranded nucleic acids can be unwound in the presence of ATP, as shown inFIG.14.

Embodiment Nine, Unwinding Experiments of E1 and its Mutant on the Artificial Lipid Membrane

After verifying the helicase activity of E1 in vitro, unwinding experiments on the artificial lipid membrane is planned: after insertion of E1 into the BLM, dsDNA could not be translocation through the E1 protein at the room temperature (21° C.) in the absence of ATP and Mg2+because of the diameter of double-stranded DNA is larger than the diameter of E1 protein pore (as mentioned above).

However, by controlling the appropriate system environment, including the concentration of ATP, Mg2+concentrations, and system temperature, dsDNA translocation through E1 is observed at the molecular level (as shown inFIG.15). As a comparison, we chose another MspA without helicase activity (the diameter of its pore is similar to that of E1 and smaller than that of double-stranded DNA) to transfer double-stranded DNA, and the results show that under the conditions of non-unwinding system (ATP-free, Mg2+) and the same temperature, ATP concentration and Mg2+concentration as E1-2, double-stranded DNA cannot be transported through MspA porin.

Because low salt concentration is needed in E1 helicase activity determination experiments, PBS is chosen to conduct the unwinding on the artificial lipid membrane; in this buffer solution, the conductance of E1-2 is about 0.2±0.02 nS.

The scheme of this experiment is described below: After BLM is formed in PBS conductance buffer, E1-2 steady inserts into BLM (as described above); then dsDNA (final concentration of 12.5 nM) is added into cis-chamber and heats up the conductance buffer with a heating plate to 37° C.; then ATP (final concentration of 5 mM) and Mg2+(final concentration of 5 mM) are added into the conductance buffer.

The result displays that, with ATP hydrolysis, dsDNA is unwound as ssDNA by E1-2 embedded in the BLM and is translocated from cis- to trans-side. Under −40 mV, amounts of dsDNA unwinding phenomenon are observed and pores will keep blockage as the voltage is reversed to +40 mV. However, with voltage changing from +40 mV to −50 mV, the unwinding occurs again and it restarts from the blocked state (as shown inFIG.16).

dsDNA with Different Lengths Unwinding on Membrane:

Moreover, the invention explores the unwinding phenomenon of dsDNA with different lengths on the artificial lipid membrane.

Recombinant plasmid PET-28b+MspA is digested with XhoI and EcoO109I to five different lengths of fragments; Since both enzymes can digests linear double strands with sticky ends, dsDNA has an extended single arm (the length is 5 bp, for the reasons mentioned above), so dsDNA with 5 different lengths is used to explore the influence of different strand's lengths on unwinding on the membrane, and the experimental process is the same as above

The results show that, under the same other conditions, with the increase of strand's length, the unwinding time of E1-2 also increases proportionally. As shown inFIG.17, under −100 mV, Ipore=−400 pA (N=20), and when the length of dsDNA is less than 2000 bp, the velocity of unwinding is relatively constant, but, with the length of dsDNA getting longer, it is found that the unwinding activity of E1-2 decreases, and when the length is close to 5000 bp, the unwinding time reaches 9 s.

dsDNA Unwinding on Membrane at Different Temperatures:

On the other hand, this invention attempts to observe the unwinding phenomenon of dsDNA on the membrane under same conditions except different temperatures. The experiments of 24 bp nucleic acid strand mentioned above are conducted at gradient temperatures, respectively, room temperature (21° C.), lower than 35° C., 35° C. −37° C., and higher than 37° C.,

The results show that the frequency of unwinding increases proportionally with the increase of temperature, but when the temperature is higher than 37° C., the unwinding begins to decrease, which is consistent with the characteristics of E1-2 as a viral helicase (as shown inFIGS.18and19).

dsDNA Unwinding on Membrane at Different Transmembrane Voltages:

This invention also determines the time required for E1-2 unwinding the same dsDNA substrate at different transmembrane voltages, and the experimental process is the same as above mentioned.

The experimental results show that with nucleic acid substrate mixing into the cis-chamber, the unwinding time is extended accordingly when the absolute voltage value is gradually reduced at negative voltage, and a non-linear function is fitted with the voltage gradient and unwinding time. The resulting curve is oriented towards the axis, which shows and proves that in addition to the driving force of voltage, E1-2 is also actively unwinding, and the direction of the two forces is the same. As shown inFIG.20, for the same dsDNA substrate, it takes about 3 s for unwinding at a voltage of −100 mV, while at a voltage of −20 mV, the unwinding time increases to nearly 30 s. According to the direction of the fitting curve, the direction of voltage-driven unwinding is the same as the direction of E1-2 unwinding. Since E1 is a mushroom structure and a single strand enters the protein pore from the opening of N-terminal during DNA replication, thus it can be known that the N-terminal opening of the protein in this state towards the negative electrode of cis-chamber.

The Unwinding of Special Nucleic Acid Substrate on Membrane:

This invention also designs another special nucleic acid substrate with three dspacers (without bases) in one strand of dsDNA, which has single arm and consists of A and T. The unwinding experiment is conducted with this special nucleic acid substrate.

It founds that in normal unwinding signal, small steps with a size of 20-30 pA appears at specific locations. And the specific locations of small step signals are consistent with those of the dspacers in nucleic acid.

Verifying E1 Unwinding on Membrane (Depending on the Function of Mg2+and ATP):

To verify the unwinding of E1-2 on membrane, excess EDTA which chelated with Mg2+is applied to inactivate the helicase. The experimental process is the same as the above mentioned. In the process of unwinding, excess of EDTA chelator is added to the reaction system.

The results show that with the increase of EDTA, the unwinding activity is gradually inhibited, and the maximum inhibition efficiency reaches to more than 80%. When excess Mg2+is added to the reaction system, it is clearly observed that the unwinding reappears, recovering to maximum efficiency by ½ or more.

At the same time, when ATPαS, another analog of ATP, is used to competitively replace ATP in the unwinding system, it is found that ATPαS could not hydrolyze to supply energy like ATP, the unwinding activity on membrane is significantly inhibited. Because this inhibition is not as reversible as EDTA chelating Mg2+, when excess ATPαS is added, the unwinding activity on membrane gradually disappears.

Studying the unwinding of E1 mutants on artificial lipid membranes with the same experimental methods and conditions, it is found that the translocation of mutant of E1 protein is initiated by 2 mM ATP. The unwinding signal similar to WT-E1 (306-605) is observed at a bias voltage of −100 mV, as shown inFIGS.27,28.

The above results show that, for the first time, a cyclic hexameric helicase of bovine papilloma virus has been successfully engineered into a protein nanopore, which is embedded in lipid bilayer membrane and polymer membrane. The Nanopore can stably stay on lipid bilayer without gating at high voltage. And it still keeps the characteristics of a helicase on artificial lipid membranes, such as temperature specificity, and competitive inhibition by ATP analogues. This nanopore can passively translocate single-stranded RNA as well as single-stranded DNA on membrane. And dsDNA with a single arm can be unwound on membrane using energy supplied by ATP hydrolysis, with the unwinding time positively proportional to the length of substrate.

The above experiments show that this invention innovatively discovers two proteins E1-1 (306-577) and E1-2 (306-605), wherein the former can be used as a new simple membrane containing conductive pore, and it can insert into lipids bilayer stably and translocation nucleic acid, the latter further has dual effects of nanopore and helicase. This invention constructs a sensing system combining the characteristics of nanopore and helicase, which replaces the existing sensing method requiring helicase a coupling nanopore, and has a good application prospect.

Further, this invention also creatively studies the mutant of E1-2 protein, obtains mutants of E1-2. It is found that the nanopore constructed by mutated monomer is more likely to capture nucleic acids and other negatively charged small molecules than the wild type protein. In addition, the mutated protein shows an increase in current range, which makes the inner cavity of the pore narrower than that of the wild type, so that the current detection of the pore is more extensive and sensitive.