CN109490579A - Magnetic field is studied to the method for DNA two dimension origami structure stability influence - Google Patents
Magnetic field is studied to the method for DNA two dimension origami structure stability influence Download PDFInfo
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- G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of research magnetic fields to the method for DNA two dimension origami structure stability influence, by the change of magnetic field strength locating for DNA paper folding solution, the method for observing the integrality of the method and fluorescence analysis of its appearance structure its double-strand.The present invention is based on scanning probe microscopy observations, and simple to operation, pattern is clear；Double-strand bonding situation sensitivity for analysis based on sepectrophotofluorometer is high and result is reliable；Interface is constructed using mica sheet, it is easy to accomplish, and the factor that can be probed into is more, transplantability is strong；Being also applied for other can be used the analysis of fluorescent dye materials；This method is also applied for probing into influence of the magnetic field to the planar materials of the pluggable fluorescent molecule of a variety of two dimensions.
Description
Technical field
The present invention is based on the characterizing methods of atomic force microscope under atmosphere and sepectrophotofluorometer, disclose a kind of research
Method of the magnetic field to DNA two dimension origami structure stability influence.Specifically related to magnetic field locating for change DNA triangle origami structure
Power simultaneously maintains certain time, and the variation of its pattern and double-strand are analyzed then in conjunction with atomic force microscope and sepectrophotofluorometer
Between bonding situation.The invention belongs to nanometer detection fields.
Background technique
DNA paper folding art be at present it is popular it is a kind of prepare two dimension or three dimensional DNA nanostructure method, only need by
After the short chain of DNA of the single-stranded and a series of partial sequence complementarity of one long DNA proportionally mixes, according to certain annealing journey
Sequence annealing.And the shape of synthesized nanostructure is easy to edit, it is only necessary to the sequence of the short chain of addition is changed, and
The sequence of short chain can be obtained by Computerized Editing, very easy.In addition to this, it is easy on DNA structure through sulfydryl, amine
Base, different large biological molecules are to realize different performances in the modifications such as carboxyl.And due to its be in organism it is inherently existing
Substance, so have brilliant biocompatibility.
The preparation that DNA origami structure is widely used in pharmaceutical carrier due to the above advantage (Chinese invention patent: utilizes
The method of DAPI insertion and release DNA nanometers of origami structures of simulation as pharmaceutical carrier, publication number: CN105004703A), biology
Sensor (Chinese invention patent: DNA paper folding nanostructure-nanogold biosensor and its system based on aptamers modification
Preparation Method and application, publication number: CN104962615A), nanometer detection (Chinese invention patent: 17 β-based on DNA nanostructure
Estradiol visible detection method and detection kit, publication number: CN104975079A) etc. fields.And DNA origami structure nanometer
The addressability of grade, the characteristics such as are modified in controllable arrangement and any position, and impart it as in terms of nanometric circuit
The unlimited imagination, therefore there is researcher to have begun trial and guide particle assemble nanometer conducting wire (Chinese invention patent: DNA with DNA
Guided nano granule assembles the preparation method of vertical type wire, publication number: CN1994863A), and started to design using DNA
Logic circuit research (Chinese invention patent: a kind of design method of the phototonus logic circuit based on DNA double helical structure,
Publication number: CN106027033A).
The application of these above-mentioned DNA paper foldings, is built upon on the basis of the integrality of this body structure of paper folding, therefore, domestic
Outer worker has extensively studied the stability of origami structure under various conditions, it is found that it is immersing ethyl alcohol, acetone etc. is organic
Structural intergrity can be kept in solvent, and in cell pyrolysis liquid then can recurring structure destruction.Although for paper folding knot
The research of the stability of structure is very detailed, but does not have research to be related to origami structure yet at present and stablize under magnetic field environment
Property, and in terms of this is applied to the influence or nanometric circuit of the targeted therapy, magnetic therapy of tumour for human body for origami structure
Using suffering from very important meaning.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of that present invention to provide a kind of research magnetic field is steady to DNA two dimension origami structure
The method of qualitative effect.
The object of the invention passes through following proposal and realizes: a kind of research magnetic field is to DNA two dimension origami structure stability influence
Method changes the power of magnetic field locating for DNA two dimension origami structure and maintains certain time using DNA two dimension origami structure as substrate,
Analyzed then in conjunction with atomic force microscope and sepectrophotofluorometer its pattern variation double-strand between bonding situation, including with
Lower step:
(1) change of environmental magnetic field intensity
The change of environmental magnetic field designs: being placed in High Field Facility containing DNA two dimension origami structure solution, (such as the Chinese Academy of Sciences is strong
Magnetic-field test device (SHMFF)) in carry out magnetization treatment, the magnetic field for changing the DNA two dimension origami structure solution present position is strong
Degree forms magnetic field gradient and changes and maintain 0.5h-70h, wherein magnetic field strength 0T-27T, gradient 0T/m-180T/m；
(2) for DNA two dimension paper folding morphology observation
Interface is constructed with mica sheet, takes 3 magnetic fields μ L treated that DNA two dimension origami structure solution is added drop-wise to the mica sheet newly removed
3 min of upper deposition, fall the salinity on interface with ultrapure water, are dried with nitrogen and are finally placed under atmosphere under scanning probe microscopy
Observation, the instrument of the model Moltimode Nanoscope VII of microscope model Veeco company, the imaging pattern
For tapping-mode.The scanning probe microscopy of other models also can be used for the observation of pattern, result will not be generated real
Matter influences；
(3) origami structure fluorescence signal is observed
18 magnetic fields μ L are taken treated DNA two dimension origami structure solution, 77 μ 1 × TAE of L Mg are added thereto2+Solution,
It adds at least 5 μ L fluorescent dyes to be transferred to after mixing in microcolorimetric ware, is placed under sepectrophotofluorometer and observes.
The synthesis of the DNA two dimension paper folding is in the steps below:
208 staple chains are equally dissolved into ultrapure water, make 200 nM of ultimate density of every chain, by M13mp18
Single stranded DNA (100 nM) and mixed short chain (200 nM) are blended in 1xTAE-Mg with the ratio of molar concentration rate 1:102+Solution
In, wherein the ultimate density of M13mp18 single stranded DNA is 5 nM, and short chain ultimate density is 50 nM, and mixed solution is put into
In PCR instrument, the setting extent of reaction is from 95 DEG C of slow coolings to 4 DEG C, and rate of temperature fall is 0.2 DEG C/10s, wherein 1xTAE-Mg2+
The ingredient of solution are as follows: 40 mM tri- (methylol) aminomethanes (Tris), 20 mM acetic acid (Acetic Acid), 2 mM ethylenediamines
Tetraacethyl (EDTA), 12.5 mM magnesium acetates (Magnesium acetate), pH 8.0.
Wherein, the long-chain is M13 phage single-chain, 7249 bp；The sequence of staple chain used is the paper folding of DNA triangle
The sequence of sequence, i.e., staple chain used is entitled " the Folding DNA to create being published on Nature in 2006
Nanoscale shapes and patterns " triangle paper folding sequence (Supporting Information, page 41).
On the basis of above scheme, change magnetic field strength locating for DNA origami structure by changing instrument setting, will,
By DNA two dimension paper folding concentration be 1nM solution be placed under the magnetic field environment of different gradients and maintain 30 min(notes: solution
Magnetic field strength locating for the amount DNA origami structure to avoid different location few as far as possible is different), it then takes out, observes as early as possible.
On the basis of above scheme, in step (3), fluorescent dye used is Eva Green dyestuff, is had than other nucleic acid
The higher sensitivity of dyestuff, and there is no the defect of " dyestuff redistribution ".To in treated in a certain amount of magnetic field paper folding solution plus
Enter enough Eva Green fluorescent dyes, is uniformly mixed and is placed in cuvette, then observed under sepectrophotofluorometer.
Eva Green dyestuff is a kind of stabilization, the DNA fluorescent dye with high sensitivity, when it is incorporated between DNA double chain,
Fluorescence signal will increase 100 times or more.The F-7000 of sepectrophotofluorometer model Hitachi company used.
On the basis of above scheme, change the magnetic field strength of DNA two dimension origami structure solution present position, magnetic field strength is
15T-27T, gradient 45T/m-180T/m；Change the magnetic field strength of paper folding solution present position and holds time as 0.5h-
70h。
The present invention is based on scanning probe microscopy under atmosphere and sepectrophotofluorometers, disclose a kind of research magnetic field pair
The experimental method of DNA two dimension origami structure stability influence, and in particular to apply varying strength to DNA triangle origami structure
Magnetic field environment observes the bonding situation between its respective pattern variation and double-strand.This method is equally applicable to other two dimensions can
It is inserted into the observation of the material of fluorescent molecule.
The present invention has the advantages that
(1) the invention proposes a kind of experimental methods of influence to DNA two-dimensional nanostructure of probing into magnetic field.
(2) observation based on scanning probe microscopy is easy to operate, and pattern is clear.Interface is constructed using mica sheet, is easy to
It realizes, and the factor that can be probed into is more, transplantability is strong.
(3) contextual analysis operation based on sepectrophotofluorometer is easy, it is strong with double-strand bonding situation correspondence, accurately
Property is good.
(4) this method is suitable for probing into influence of the magnetic field to the planar materials of the pluggable fluorescent molecule of a variety of two dimensions.
Detailed description of the invention
Fig. 1 is DNA triangle origami structure in 15 T, the magnetic field strength of 135 T/m treated shape appearance figure；
Fig. 2 is fluorescence analysis figure of the DNA triangle origami structure under different magnetic field intensity.
Specific embodiment
Technical solution of the present invention is described further below by way of specific embodiment.Embodiment below is to this
The further explanation of invention, and do not limit the scope of the invention.
Material prepares: the synthesis of DNA triangle origami structure:
208 staple chains are equally dissolved into ultrapure water, make 200 nM of ultimate density of every chain, by M13mp18
Single stranded DNA (100 nM) and mixed short chain (200 nM) are blended in 1xTAE-Mg with the ratio of molar concentration rate 1:102+Solution
In, wherein the ultimate density of M13mp18 single stranded DNA is 5 nM, and short chain ultimate density is 50 nM, and mixed solution is put into
In PCR instrument, the setting extent of reaction is from 95 DEG C of slow coolings to 4 DEG C, and rate of temperature fall is 0.2 DEG C/10s, wherein 1xTAE-Mg2+
The ingredient of solution are as follows: 40 mM tri- (methylol) aminomethanes (Tris), 20 mM acetic acid (Acetic Acid), 2 mM ethylenediamines
Tetraacethyl (EDTA), 12.5 mM magnesium acetates (Magnesium acetate), pH 8.0.
Wherein, the long-chain is M13 phage single-chain, 7249 bp；The sequence of staple chain used is the paper folding of DNA triangle
Sequence is entitled " the Folding DNA to create nanoscale shapes and being published on Nature in 2006
Patterns " triangle paper folding sequence (Supporting Information, page 41).
Embodiment 1
At Chinese Academy of Sciences's High Field Facility (SHMFF)) in triangle paper folding solution (1nM) carry out magnetization treatment, magnetic field strength
15 T, 135 T/m of gradient, the morphology observation of DNA triangle paper folding after magnetic field strength processing:
Mica sheet is fixed on iron plate, processed triangle paper folding solution (1nM) under 3 magnetic fields μ l is taken to deposit to new removing mica
On piece washes away salinity and is dried with nitrogen, and is placed under scanning probe microscopy and observes the variation of its pattern, as shown in Figure 1, DNA triangle is rolled over
Paper is the triangle of rule, and three angles are sharp keen visible.It is 15T when being placed on magnetic field strength, magnetic field gradient is 135T/m's
In magnetic field after 30 minutes, shape changes, and triangular structure is disintegrated.Visible irregular particle shape DNA in figure disintegrates for paper folding
Aggregation afterwards, part triangle has still maintained fuzzy shape, but its visible height of height map is got higher, and is illustrating its shape just
During gradually disintegrating.
The fluorescence analysis of DNA triangle paper folding after the magnetic field strength processing of 15 T, 135 T/m:
18 μ l are taken treated paper folding solution, 77 μ 1 × TAE of l Mg are added thereto2+Solution, be added 5 μ l 20 ×
Eva Green dyestuff, is transferred to after mixing in microcolorimetric ware, is placed under sepectrophotofluorometer and observes, such as Fig. 2 institute
Show, abscissa line is magnetic field strength (T), magnetic field gradient (T/m).After ordinate is placed in magnetic field 30 minutes for DNA origami structure,
Using fluorescence spectrum, enough Eva Green fluorescent dyes are added in DNA structure for the fluorescent absorption value measured, and this dyestuff is only
Having can just shine when DNA structure keeps complete.Therefore absorption value is bigger, shows that DNA structure is more complete.It is magnetic field strength in figure
With the coefficient result of gradient.
Embodiment 2
It is approximate with embodiment 1, only 26.8 T of magnetic field strength, 45 T/m of gradient, the shape of DNA triangle paper folding after magnetic field strength processing
Looks observation:
Mica sheet is fixed on iron plate, processed triangle paper folding solution (1 nM) under 3 magnetic fields μ l is taken to deposit to new removing cloud
Salinity is washed away on master slice and is dried with nitrogen, and is placed under scanning probe microscopy and is observed the variation of its pattern.
The fluorescence analysis of DNA triangle paper folding after the magnetic field strength processing of 26.8 T, 45 T/m:
18 μ l are taken treated paper folding solution, 77 μ 1 × TAE of l Mg are added thereto2+Solution, add 5 μ l 20 ×
Eva Green dyestuff, is transferred to after mixing in microcolorimetric ware, is placed under sepectrophotofluorometer and observes, such as Fig. 2 institute
Show.
Embodiment 3
It is approximate with embodiment 1, DNA triangle paper folding after the magnetic field strength processing of only 20.5 T of magnetic field strength, 180 T/m of gradient
Morphology observation:
Mica sheet is fixed on iron plate, processed triangle paper folding solution (1nM) under 3 magnetic fields μ l is taken to deposit to new removing mica
On piece washes away salinity and is dried with nitrogen, and is placed under scanning probe microscopy and observes the variation of its pattern.
The fluorescence analysis of DNA triangle paper folding after the magnetic field strength processing of 20.5 T, 180 T/m:
18 μ l are taken treated paper folding solution, 77 μ 1 × TAE of l Mg are added thereto2+Solution, be added 5 μ l 20 ×
Eva Green dyestuff, is transferred to after mixing in microcolorimetric ware, is placed under sepectrophotofluorometer and observes, such as Fig. 2 institute
Show.
Claims (6)
1. a kind of research magnetic field is to the method for DNA two dimension origami structure stability influence, using DNA two dimension origami structure as substrate,
It is characterized in that changing the power of magnetic field locating for DNA two dimension origami structure and maintaining certain time, then in conjunction with atomic force microscopy
Mirror and sepectrophotofluorometer analyze bonding situation between the variation of its pattern and double-strand, comprising the following steps:
(1) change of environmental magnetic field intensity
The change of environmental magnetic field designs: will be placed in High Field Facility (SHMFF) containing DNA two dimension origami structure solution into
Row magnetization treatment, the magnetic field strength for changing the DNA two dimension origami structure solution present position form magnetic field gradient and change and maintain
0.5h-70h, wherein magnetic field strength 0T-27T, gradient 0T/m-180T/m；
(2) for DNA two dimension paper folding morphology observation
Interface is constructed with mica sheet, takes 3 magnetic fields μ L treated that DNA two dimension origami structure solution is added drop-wise to the mica sheet newly removed
3 min of upper deposition, fall the salinity on interface with ultrapure water, are dried with nitrogen and are finally placed under atmosphere under scanning probe microscopy
Observation；
(3) origami structure fluorescence signal is observed
18 magnetic fields μ L are taken treated DNA two dimension origami structure solution, 77 μ 1 × TAE of L Mg are added thereto2+Solution, then
At least 5 μ L fluorescent dyes are added to be transferred to after mixing in microcolorimetric ware, is placed under sepectrophotofluorometer and observes.
2. method of the research magnetic field to DNA two dimension origami structure stability influence as described in claim 1, which is characterized in that institute
State the synthesis of DNA triangle paper folding in the steps below:
208 staple chains are equally dissolved into ultrapure water, make 200 nM of ultimate density of every chain, by M13mp18
Single stranded DNA (100 nM) and mixed short chain (200 nM) are blended in 1xTAE-Mg with the ratio of molar concentration rate 1:102+Solution
In, wherein the ultimate density of M13mp18 single stranded DNA is 5 nM, and short chain ultimate density is 50 nM, and mixed solution is put into
In PCR instrument, the setting extent of reaction is from 95 DEG C of slow coolings to 4 DEG C, and rate of temperature fall is 0.2 DEG C/10s, wherein 1xTAE-Mg2+
The ingredient of solution are as follows: 40 mM tri- (methylol) aminomethanes (Tris), 20 mM acetic acid (Acetic Acid), 2 mM ethylenediamines
Tetraacethyl (EDTA), 12.5 mM magnesium acetates (Magnesium acetate), pH 8.0.
3. method of the research magnetic field to DNA two dimension origami structure stability influence as claimed in claim 2, which is characterized in that institute
Stating long-chain is M13 phage single-chain, 7249 bp；The sequence of staple chain used is DNA triangle paper folding sequence.
4. method of the research magnetic field to DNA two dimension origami structure stability influence as described in claim 1, which is characterized in that logical
It crosses and changes instrument setting to change magnetic field strength locating for DNA origami structure, the DNA two dimension origami structure for being 1nM by concentration
Solution is placed under the magnetic field environment of different gradients and maintains 30 min.
5. method of the research magnetic field to DNA two dimension origami structure stability influence as described in claim 1, which is characterized in that step
Suddenly in (3), fluorescent dye used is Eva Green dyestuff.
6. studying magnetic field as described in claim 1 or 4 to the method for DNA two dimension origami structure stability influence, which is characterized in that
Change the magnetic field strength of paper folding solution present position, magnetic field strength 15T-27T, gradient 45T/m-180T/m.
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| CN115651929A (en) * | 2022-11-18 | 2023-01-31 | 中国科学院合肥物质科学研究院 | Separation method based on DNA magnetic difference |
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