CN113151604A - PNA probe structure for detecting novel coronavirus and synthetic method thereof - Google Patents
PNA probe structure for detecting novel coronavirus and synthetic method thereof Download PDFInfo
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Abstract
The invention provides a PNA probe structure for detecting novel coronavirus and a synthetic method thereof, and particularly relates to the technical field of viruses. The invention comprises the following steps: synthesizing peptide nucleic acid monomer-resin; synthesizing an oligomer-resin; linkage of the 5' end fluorophore. The PNA probe structure and the synthetic method for detecting the novel coronavirus can improve the detection speed, sensitivity and accuracy of the novel coronavirus.
Description
Technical Field
The invention belongs to the technical field of viruses, and particularly relates to a PNA probe structure for detecting a novel coronavirus and a synthetic method thereof.
Background
All organisms, except prions, contain nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). A novel coronavirus is a virus that contains only RNA, and the specific RNA sequence in the virus is a marker, i.e. a target sequence, that distinguishes the virus from other pathogens.
The most common method for detecting specific sequences of novel coronaviruses is fluorescence quantitative PCR (polymerase chain reaction). The reagent for detecting nucleic acid is actually a specific oligonucleotide sequence which is matched with a specific RNA target sequence in the virus, and the specific oligonucleotide sequence and the specific RNA target sequence release a fluorescent signal after hybridizing through a base pairing principle, so that the virus is specifically identified.
Among many nucleic acid probes, fluorescent nucleic acid probes mainly use fluorescence intensity as a signal for quantitative analysis, have the characteristics of high sensitivity, design diversification, strong quantitative analysis capability and the like, and become one of research hotspots of analytical chemistry. However, the fluorescent nucleic acid probe also has the defects of large background noise, not ideal recognition specificity, false positive signals and the like.
PNA is an artificially synthesized DNA analog with a polypeptide backbone, and because of its structure similar to DNA and its similarity, PNA is capable of hybridizing to single stranded nucleic acids via Watson-Crick base complementary forms to form stable complexes of duplexes. More importantly, the affinity of PNA with DNA and RNA is stronger than that of DNA, the hybridization stability is greatly improved, one base mismatch cannot be allowed in Watson-Crick principle, the specificity is also greatly improved, and in addition, the PNA has good resistance to nuclease and protease. Based on the three points, the PNA peptide nucleic acid probe has better sensitivity, stronger stability and higher selectivity, and has more development significance.
Nucleic acid probe detection is a major and important test means for diagnosis, but the bottleneck is that the detection speed, detection sensitivity and detection accuracy (or specificity or selectivity) need to be improved. Therefore, there is a need for a PNA probe structure and synthesis method for detecting novel coronavirus that can solve the above problems.
Disclosure of Invention
The invention aims to provide a PNA probe structure for detecting novel coronavirus and a synthesis method thereof, which can improve the detection speed, sensitivity and accuracy of the novel coronavirus.
The invention provides the following technical scheme:
a PNA probe structure for detecting novel coronavirus is characterized in that peptide nucleic acid (namely PNA) is used as a skeleton for simulation and replacement of a pentose phosphate skeleton in conventional DNA or RNA, namely the probe skeleton is used as a PNA skeleton; the correct sequence of base sequences on the PNA side chains for hybridization recognition is the following sequence: 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3' or 5 '-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'.
A synthetic method of a PNA probe structure for detecting novel coronavirus, a method for preparing 0.1mmol PNA comprises the following steps:
s1: adding 0.1g of resin and a proper amount of DMF into a column reactor, shaking for swelling for 30min, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving Fmoc-peptide nucleic acid monomer 0.5mmol, DCC0.5mmol, HOSU 0.5mmol and DMAP0.1mmol with appropriate amount of DMF, transferring to a solid phase reactor after half an hour, adding appropriate amount of DMF, shaking for 3h in dark place, draining after the reaction is finished, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and draining;
s2: adding a deprotection reagent 20% (V/V) piperidine/DMF solution into the solution prepared in S1, shaking for 30min to remove the protecting group Fmoc of the first PNA monomer on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of second Fmoc-PNA monomer with the base sequence of 3'→ 5', 0.3mmol of DCC, 0.3mmol of HOSU and 0.1mmol of DMAP with a proper amount of DMF, transferring to a solid phase reactor filled with peptide nucleic acid-resin after half an hour, adding a proper amount of DMF, and shaking away from the sun for reaction for 3 hours; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping; repeating the steps in S2, removing the Fmoc protecting group at the N-terminal, entering the cycle of connecting the next peptide nucleic acid monomer, and continuously condensing two by two until the expected oligomer is synthesized; after the final condensation, the obtained oligomer-resin was washed with methanol 5 times, shaken for 2min each time, pumped, vacuum dried overnight, and weighed;
s3: weighing a certain amount of oligomer-resin compound prepared in the step S2, putting the oligomer-resin compound into a reactor, preparing a cutting reagent according to a proportion, and putting the oligomer-resin compound into a refrigerator to cool for several minutes; slowly adding a cutting agent into the reactor, and magnetically stirring for 3 hours at room temperature; filtering, adding 4ml of cracking solution, and repeatedly cutting for 0.5 h; filtration, washing the resin with 10ml 90% TFA/DCM, transferring the washings together with the filtrate into a flask, and distilling under reduced pressure at 35 ℃; dropwise adding ten times of volume of pre-cooled diethyl ether into the concentrated solution, oscillating, and standing in a refrigerator for 30 min; centrifuging at high speed, removing supernatant, and vacuum drying overnight to obtain crude product; dissolving with redistilled water, lyophilizing to obtain white powder, and weighing.
The linkage of the 5' end fluorophore comprises the following steps:
s1: the synthesis of FAM-PNA is carried out,
adding a deprotection reagent 20% (V/V) piperidine/DMF solution, shaking for 30min to remove the Fmoc protecting group on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of FAM (5-carboxyfluorescein), 0.3mmol of DCC, 0.3mmol of HOSU and 0.1mmol of DMAP in proper amount of DMF, transferring to a solid phase reactor filled with PNA-resin, adding proper amount of DMF, and shaking for 3h in the dark; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping;
s2: FAM-PNA-TAMRA synthesis,
dissolving TAMRA (rhodamine) 0.3mmol, DCC 0.3mmol, HOSU 0.3mmol and DMAP0.1mmol in DMF, adding into crude PNA cut in S1, and shaking for 3 hr in the dark; adding water to quench after the reaction is finished, performing reduced pressure spin-drying, adding diethyl ether, shaking and washing, and standing in a refrigerator for 30 min; centrifuging at high speed, carefully pouring out the supernatant, and vacuum drying overnight to obtain crude product; dissolving with redistilled water, and lyophilizing to obtain white powder, i.e. FAM-PNA-TAMRA.
Preferably, 0.5mmol of Fmoc-peptide nucleic acid monomer in S1 is the peptide nucleic acid monomer of the first base from the 3' end of the base sequence; the PNA monomers in S2 were sequentially extended 3'→ 5' one by one.
Preferably, the free amino group of the condensation reaction is measured by ninhydrin color development in step S2 to determine whether the reaction has proceeded completely.
Preferably, tens of bases-resin complexes prepared in step S2 are taken out and placed in a test tube, and DMF is added to wash the tubes for 3 times; adding two drops of ninhydrin solution respectively; heating at 120 deg.C for 3 min; the color of the resin was observed.
Preferably, in the step S2, 5g of ninhydrin is dissolved in 100ml of ethanol, and the solution is promoted by slight heating or stirring.
Preferably, the resin in step S1 is 1 mmol/g.
Preferably, 5ml of DMF is added in total in steps S1 and S2.
Preferably, the cleavage reagent TFA in step S3 EDT anisole 38:1:1 for 3.5h at 26 ℃ and diethyl ether to precipitate the crude peptide.
The invention has the beneficial effects that:
the PNA is different from DNA and RNA with negative charges, the skeleton of the PNA is neutral, electrostatic repulsion does not exist between the PNA and the DNA and the RNA, and the stability of combination is greatly improved;
the backbone of PNA is different from that of DNA/RNA, but the combination of PNA and DNA/RNA still strictly obeys the Watson-Crick principle, a hybrid chain can be formed more quickly by using a shorter sequence (12-15 mer), and single base mismatch can be identified, so that the detection speed, sensitivity and accuracy of the novel coronavirus can be improved;
PNA hardly reacts with protein, so that toxic effect is small;
the hybridization of PNA with DNA or RNA is hardly affected by the salt concentration of the hybridization system;
the high sensitivity and specificity of the PNA probe are applied to a detection method of microorganisms or viruses, the pretreatment of a sample is very similar to the traditional detection technology, and an exact result can be directly obtained without other detections;
compared with DNA synthesis, PNA synthesis difficulty is low, and yield is high;
drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of PNA backbone synthesis of the present invention;
FIG. 2 is a schematic illustration of base acetylation according to the present invention;
FIG. 3 is a schematic diagram of PNA monomer synthesis of the present invention;
FIG. 4 is a schematic diagram of the synthesis of PNA oligomers of the present invention.
Detailed Description
As shown in FIGS. 1 to 4, a PNA probe structure for detecting novel coronavirus uses peptide nucleic acid (i.e., PNA) as a backbone to mimic and replace the pentose phosphate backbone in conventional DNA or RNA, i.e., the probe backbone is a PNA backbone; the correct sequence of base sequences on the PNA side chains for hybridization recognition is the following sequence: 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3' or 5 '-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'.
A synthetic method of a PNA probe structure for detecting novel coronavirus, a method for preparing 0.1mmol PNA comprises the following steps:
s1: adding 0.1g of 1mmol/g resin and a proper amount of DMF into a column reactor, shaking for swelling for 30min, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving Fmoc-peptide nucleic acid monomer 0.5mmol (peptide nucleic acid monomer of the first base at 3' end of base sequence highlighted in PNA probe structure), DCC0.5mmol, HOSU 0.5mmol, and DMAP0.1mmol with appropriate amount of DMF, transferring to solid phase reactor after half an hour, adding appropriate amount of DMF (5 ml in total), shaking away from light for 3h, draining after reaction, washing with DMF, DCM, and MeOH for 3 times, shaking for 3min each time, and draining.
S2: adding a deprotection reagent 20% (V/V) piperidine/DMF solution into the solution prepared in S1, shaking for 30min to remove the Fmoc protecting group on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of second Fmoc-PNA monomer with base sequence 3'→ 5', 0.3mmol of DCC, 0.3mmol of HOSU, and 0.1mmol of DMAP with appropriate amount of DMF, transferring to a solid phase reactor containing peptide nucleic acid-resin after half an hour, adding appropriate amount of DMF (total 5ml), and shaking away from the sun for reaction for 3 h; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping;
the method comprises the following steps of determining free amino of condensation reaction by a ninhydrin color development method, judging whether the reaction is complete or not, wherein the principle is that the free alpha-amino and ninhydrin are heated together to cause amino acid oxidative deamination and decarboxylation, and then ninhydrin reacts with reaction products (ammonia) and reduced ninhydrin to generate a purple substance (DYDA); putting dozens of basic groups-resin complexes prepared in the step S2 into a test tube, and adding DMF to wash for 3 times; adding two drops of ninhydrin solution (5g ninhydrin dissolved in 100ml ethanol, slightly heating or stirring to promote dissolution); heating at 120 deg.C for 3 min; observing the color of the resin; the colorless state shows that the free ammonia on the resin is completely connected with the next Fmoc-base, and the next operation can be carried out; if the resin is bluish purple, the connection reaction is incomplete, and the reaction time is prolonged until the resin is colorless, so that the next reaction can be carried out;
repeating the above steps in S2, removing the Fmoc protecting group from the N-terminus, entering a cycle of connecting the next peptide nucleic acid monomer (PNA monomer sequence PNA probe structure emphasizing sequence 3'→ 5' one-by-one elongation), and condensing two-by-two continuously until the desired oligomer is synthesized; after the final condensation, the oligomer-resin obtained was washed with methanol 5 times, shaken for 2min each time, drained, vacuum dried overnight and weighed.
S3: weighing a certain amount of oligomer-resin compound prepared in the step S2, putting the oligomer-resin compound into a reactor, and preparing a cutting reagent according to the proportion of TFA, EDT, anisole, 38:1:1 (the time is 3.5h, the temperature is 26 ℃), and putting the oligomer-resin compound into a refrigerator to cool for 10 min; slowly adding a cutting agent into the reactor, and magnetically stirring for 3 hours at room temperature; filtering, adding 4ml of cracking solution, and repeatedly cutting for 0.5 h; filtration, washing the resin with 10ml 90% TFA/DCM, transferring the washings together with the filtrate into a flask, and distilling under reduced pressure at 35 ℃; dropwise adding ten times of volume of pre-cooled diethyl ether into the concentrated solution, oscillating, and standing in a refrigerator for 30 min; centrifuging at high speed, removing supernatant, vacuum drying overnight, and precipitating with diethyl ether to obtain crude peptide; dissolving with redistilled water, lyophilizing to obtain white powder, and weighing.
The linkage of the 5' end fluorophore comprises the following steps:
s1: the synthesis of FAM-PNA is carried out,
adding a deprotection reagent 20% (V/V) piperidine/DMF solution, shaking for 30min to remove the Fmoc protecting group on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of FAM (5-carboxyfluorescein), 0.3mmol of DCC, 0.3mmol of HOSU and 0.1mmol of DMAP in proper amount of DMF, transferring to a solid phase reactor filled with PNA-resin, adding proper amount of DMF, and shaking for 3h in the dark; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping;
s2: FAM-PNA-TAMRA synthesis,
dissolving TAMRA (rhodamine) 0.3mmol, DCC 0.3mmol, HOSU 0.3mmol and DMAP0.1mmol in DMF, adding into crude PNA cut in S1, and shaking for 3 hr in the dark; adding water to quench after the reaction is finished, performing reduced pressure spin-drying, adding diethyl ether, shaking and washing, and standing in a refrigerator for 30 min; centrifuging at high speed, carefully pouring out the supernatant, and vacuum drying overnight to obtain crude product; dissolving with redistilled water, and lyophilizing to obtain white powder, i.e. FAM-PNA-TAMRA.
The PNA is different from DNA and RNA with negative charges, the skeleton of the PNA is neutral, electrostatic repulsion does not exist between the PNA and the DNA and the RNA, and the stability of combination is greatly improved; the backbone of PNA is different from that of DNA/RNA, but the combination of PNA and DNA/RNA still strictly obeys the Watson-Crick principle, a hybrid chain can be formed more quickly by using a shorter sequence (12-15 mer), and single base mismatch can be identified, so that the detection speed, sensitivity and accuracy of the novel coronavirus can be improved; PNA hardly reacts with protein, so that toxic effect is small; the hybridization of PNA with DNA or RNA is hardly affected by the salt concentration of the hybridization system; the high sensitivity and specificity of the PNA probe are applied to a detection method of microorganisms or viruses, the pretreatment of a sample is very similar to the traditional detection technology, and an exact result can be directly obtained without other detections; PNA synthesis is less difficult and has higher yield than DNA synthesis.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A PNA probe structure for detecting novel coronavirus is characterized in that: peptide nucleic acid (namely PNA) is used as a skeleton for simulating and replacing a pentose phosphate skeleton in conventional DNA or RNA, namely a probe skeleton is used as a PNA skeleton; the correct sequence of base sequences on the PNA side chains for hybridization recognition is the following sequence: 5'-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3' or 5 '-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'.
2. The method for synthesizing PNA probe structure for detecting novel coronavirus according to claim 1, wherein the method for preparing 0.1mmol PNA comprises the following steps:
s1: adding 0.1g of resin and a proper amount of DMF into a column reactor, shaking for swelling for 30min, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving Fmoc-peptide nucleic acid monomer 0.5mmol, DCC0.5mmol, HOSU 0.5mmol and DMAP0.1mmol with a proper amount of DMF, transferring to a solid phase reactor after half an hour, adding a proper amount of DMF, shaking for 3 hours in the dark, draining after the reaction is finished, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and draining;
s2: adding a deprotection reagent 20% (V/V) piperidine/DMF solution into the solution prepared in S1, shaking for 30min to remove the protecting group Fmoc of the first PNA monomer on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of second Fmoc-PNA monomer with the base sequence of 3'→ 5', 0.3mmol of DCC, 0.3mmol of HOSU and 0.1mmol of DMAP with a proper amount of DMF, transferring to a solid phase reactor filled with peptide nucleic acid-resin after half an hour, adding a proper amount of DMF, and shaking away from the sun for reaction for 3 hours; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping; repeating the steps in S2, removing the Fmoc protecting group at the N-terminal, entering the cycle of connecting the next peptide nucleic acid monomer, and continuously condensing two by two until the expected oligomer is synthesized; after the final condensation, the obtained oligomer-resin was washed with methanol 5 times, shaken for 2min each time, pumped, vacuum dried overnight, and weighed;
s3: weighing a certain amount of oligomer-resin compound prepared in the step S2, putting the oligomer-resin compound into a reactor, preparing a cutting reagent according to a proportion, and putting the oligomer-resin compound into a refrigerator to cool for several minutes; slowly adding a cutting agent into the reactor, and magnetically stirring for 3 hours at room temperature; filtering, adding 4ml of cracking solution, and repeatedly cutting for 0.5 h; filtration, washing the resin with 10ml of 90% TFA/DCM, transferring the washings together with the filtrate into a flask, and distilling under reduced pressure at 35 ℃; dropwise adding ten times of volume of pre-cooled diethyl ether into the concentrated solution, oscillating, and standing in a refrigerator for 30 min; centrifuging at high speed, removing supernatant, and vacuum drying overnight to obtain crude product; dissolving with redistilled water, lyophilizing to obtain white powder, and weighing.
3. The method for synthesizing PNA probe structure for detecting novel coronavirus according to claim 2, wherein the bonding of the 5' end fluorophore comprises the following steps:
s1: the synthesis of FAM-PNA is carried out,
adding a deprotection reagent 20% (V/V) piperidine/DMF solution, shaking for 30min to remove the Fmoc protecting group on the resin, draining, washing with DMF for 6 times, shaking for 3min each time, and draining; dissolving 0.3mmol of FAM (5-carboxyfluorescein), 0.3mmol of DCC, 0.3mmol of HOSU and 0.1mmol of DMAP in proper amount of DMF, transferring to a solid phase reactor filled with PNA-resin, adding proper amount of DMF, and shaking for 3h in the dark; after the reaction is finished, pumping, washing with DMF, DCM and MeOH for 3 times respectively, shaking for 3min each time, and pumping;
s2: FAM-PNA-TAMRA synthesis,
dissolving TAMRA (rhodamine) 0.3mmol, DCC 0.3mmol, HOSU 0.3mmol and DMAP0.1mmol in proper amount of DMF, adding into crude PNA cut in S1, and shaking for reaction for 3h in the dark; adding water to quench after the reaction is finished, performing reduced pressure spin-drying, adding diethyl ether, shaking and washing, and standing in a refrigerator for 30 min; centrifuging at high speed, carefully pouring out the supernatant, and vacuum drying overnight to obtain crude product; dissolving with redistilled water, and lyophilizing to obtain white powder, i.e. FAM-PNA-TAMRA.
4. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: 0.5mmol of Fmoc-peptide nucleic acid monomer in S1 is peptide nucleic acid monomer of the first base from the 3' end of the base sequence; the PNA monomers in S2 were sequentially extended 3'→ 5' one by one.
5. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: in the step S2, the free amino group in the condensation reaction was measured by ninhydrin color development to determine whether or not the reaction was completely carried out.
6. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: putting dozens of basic groups-resin complexes prepared in the step S2 into a test tube, and adding DMF to wash for 3 times; adding two drops of ninhydrin solution respectively; heating at 120 deg.C for 3 min; the color of the resin was observed.
7. The method for synthesizing PNA probe structure for detecting novel coronavirus according to claim 5, wherein the PNA probe structure comprises the following steps: in step S2, 5g of ninhydrin is dissolved in 100ml of ethanol, and slightly heated or stirred to promote dissolution.
8. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: the resin in the S1 step was 1 mmol/g.
9. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: a total of 5ml DMF was added in steps S1 and S2, respectively.
10. The method of claim 2, wherein the PNA probe structure is selected from the group consisting of: in step S3 the cleavage reagent TFA EDT anisole 38:1:1 was added over 3.5h at 26 ℃ and the crude peptide was precipitated with ether.
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| 王翼: ""靶向新型冠状病毒包膜蛋白的小分子抑制剂发现"", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》, no. 08, 15 August 2021 (2021-08-15), pages 079 - 41 * |
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