CN105602949A - Nucleic acid structure of which interchain exchange is achieved by support DNA (deoxyribonucleic acid) and synthesis method thereof - Google Patents

Nucleic acid structure of which interchain exchange is achieved by support DNA (deoxyribonucleic acid) and synthesis method thereof Download PDF

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CN105602949A
CN105602949A CN201610063979.6A CN201610063979A CN105602949A CN 105602949 A CN105602949 A CN 105602949A CN 201610063979 A CN201610063979 A CN 201610063979A CN 105602949 A CN105602949 A CN 105602949A
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陈瑞鹏
开明轩
崔妍
弭永利
魏迪明
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Tsinghua University
Tongji University
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Tongji University
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Abstract

The invention discloses a nucleic acid structure of which the interchain exchange is achieved by a support DNA (deoxyribonucleic acid). The nucleic acid structure is a two-dimensional or three-dimensional nucleic acid structure with controllable size and shape, which is formed by achieving interchain exchange by the support DNA. The nucleic acid structure comprises a set of parallel double helices connected by interchain exchange and a single-chain DNA segment, wherein the double helices are formed by carrying out folding and interchain exchange by the support DNA, carrying out specific matching with a plurality of short-chain DNAs and carrying out annealing reaction; and the single-chain DNA is the part of the support DNA which does not form the double helices. The invention provides the multiple nucleic acid structures and synthesis methods thereof. The nucleic acid structure is a variant technique based on DNA paper folding. The invention aims to form the nucleic acid structure with controllable size, shape, complexity and modification by achieving interchain exchange by using the support DNA. The nucleic acid structure is completed by the support DNA/short-chain DNA self-assembly process, and has the advantages of higher yield and simple design; and the predicated two-dimensional and three-dimensional structures can be simultaneously synthesized.

Description

Interchain exchanges nucleic acid structure and the synthetic method thereof reached by support DNA
Technical field
The present invention relates to nucleic acid structure and the synthetic method thereof of DNA field of nanometer technology. More particularly, relate to utilizationThe method of the two and three dimensions nucleic acid structure of the controlled size of chain that frame DNA reaches exchange synthesis of nano rank, shape, complexity andNucleic acid structure itself. Based on the addressability of every short chain DNA in resulting structures, can obtain and there is specific hole and modificationNucleic acid structure.
Background technology
Last century the eighties, it is multiple that Seeman proposes to utilize the principle of DNA base complementrity pairing DNA can be assembled into firstAssorted space structure, has started and has utilized DNA as nanoscale construction material but not the frontier of carrier of genetic information, and by its lifeDNA nanometer technology by name. Subsequently, researcher is by the different primitive modules of structure, as DX (double-crossover), TX(triple-crossover) module,, cross module and symmetrical module, and obtain graphic structure miscellaneous with module assembled(two-dimensional array, square net etc.), but agent approach assembling is to connect into larger by means of the base complementrity pairing of minor structure unitGraphic structure, its size and dimension is difficult to accurate control.
2006, Rothemund proposed a brand-new noun: " DNA paper folding art " (DNAorigami). He is by oneBar genomic DNA long-chain (M13mp18) mixes with hundreds of short chain DNA, is undertaken by the base complementrity pairing at ad-hoc locationFolding connection, has obtained triangle, five-pointed star, smiling face etc. just like the general mirable complicated two-dimensional structure of paper folding works, thanThe structure obtaining by module DNA self-assembling method is more accurate, can be rated as the once landmark breakthrough of DNA nanometer technology.
Thereafter, the achievement based on DNA paper folding art emerges in an endless stream. 2007, the people such as Williamshih were by M13mp18 supportBe folded into six rotation nanotubes, construct three-dimensional structure with DNA first, realized the breakthrough to three-dimensional structure by X-Y scheme.2009, they proposed honeycomb pleated model on the basis of nanotube, had built successively multiple monomers such as bridge-type, bottleneck and groupClose structure. In research subsequently, they have realized figure entirety distortion or bending angle again by additions and deletions base in each unitThe accurate control of degree, has pushed three dimensional DNA paper folding art to a new height.
Summary of the invention
Object of the present invention, exactly in order to provide a kind of interchain to exchange nucleic acid structure and the preparation thereof of being reached by support DNAMethod and purposes.
Object of the present invention can be achieved through the following technical solutions: a kind of interchain exchanges the core of being reached by support DNAAcid structure, is to reach by support DNA two dimension or the three-dimensional nucleic acid structure with controlled size and shape that interchain exchange forms,This nucleic acid structure comprises one group of parallel double helix and one section of single stranded DNA, and described double helix passes through folding, interchain by support DNAExchange is carried out specificity with many short chain DNA and is mated, and forms via annealing reaction, and single stranded DNA is in support DNA, not form pair spiral shellsThe part of revolving.
Described support DNA comprises the single stranded DNA of M13mp18 or other specified sequence; M13mp18 is m13 phage geneA circular single-stranded DNA in group DNA, contains 7249 nucleotides altogether, and its sequence is as shown in SEQIDNO1.
Described interchain exchange refers to support DNA and carries out in a pair of chain exchange site of adjacent double helix same positionThe behavior connecting. Multiple chain exchanges site pair is set at the every row double helix of the nucleic acid structure place of keeping at a certain distance away, support DNA'sFolding path enters the chain exchange site of adjacent double helix same position, every row via the double-helical chain exchange of every a line siteDouble-helical chain exchange site is 2 to 500 pairs, to same a line double helix, and distance (the chain exchange week between each chain exchange site pairPhase) be 4 to 416 nucleotides. Exchange the double helix of adjacent position in structure is joined to one another by interchain, all chainsBetween exchange and all reach by support DNA.
Described nucleic acid structure is two-dimentional nucleic acid structure, and this two dimension nucleic acid structure comprises 2 to 200 row double helixs, and a segment length is singleChain DNA and 20 to 1000 short chain DNA that mate with support DNA at least partly; Every row double helix comprises 60 to 6000 nucleosidesAcid, chain exchange cycle is 4 to 416; Short chain DNA follows base complementrity pair principle with mating of support DNA in nucleic acid structure,The length of every short chain DNA can equate and all be less than 400 nucleotides to same structure. For single nucleic acid structure, its bagEvery the short chain DNA containing is unique, only in structure, occurs once; For different nucleic acid structures, short chain DNA can phaseWith.
Described two-dimentional nucleic acid structure be square, polygon or other rules forming in the length restriction of support DNA orIrregular X-Y scheme; The difformity of two dimension nucleic acid structure by change the folding path of support DNA and short chain DNACoordination is put and is reached.
Described nucleic acid structure is the 3-D solid structure of multilayer nucleic acid composition, comprises segment length's single stranded DNA and 20 to 1000The short chain DNA that bar mates with support DNA at least partly, 2 to 100 layers of double helix, the spiral number that every helical layer comprises is 2 to 100OK, every row spiral comprises 60 to 3000 nucleotides, and chain exchange cycle is 4 to 416, short chain DNA in nucleic acid structure with supportThe coupling of DNA is followed base complementrity pair principle, and the length of every short chain DNA can equate same structure and all be less than 200Individual nucleotides; For single nucleic acid structure, its every short chain DNA comprising is unique, only in structure, occurs once; ForDifferent nucleic acid structures, short chain DNA can be identical.
Described three-dimensional nucleic acid structure is cube, polyhedron or other rules forming in the length restriction of support DNAOr irregular three-dimensional structure; The difformity of three-dimensional nucleic acid structure is by changing the folding path of support DNA and short chain DNAMatched position is reached.
Described nucleic acid structure comprises the nucleic acid structure with hole, and this hole is by one in eliminating nucleic acid structure or manyBar short chain DNA, make support DNA in this position not participation structure form and reach, that described hole comprises is square, polygon, crossShape and other rule or irregular pattern.
The nucleic acid that the described nucleic acid structure with hole comprises " universe " hexagram of having in the Eight Diagrams of representing and " female " hexagram holeStructure, " universe " hexagram and " female " hexagram hole are by leaving out 60 and 48 short chain nucleotides shapes of ad-hoc location in complete nucleic acid structureBecome, the short chain nucleotides of being left out does not participate in annealing process.
Described nucleic acid structure is included in single specific site in multiple range of structures and has the nucleic acid knot of chemical modificationStructure, the nucleic acid with chemical modification directly as short chain DNA or indirectly by with short chain DNA not with the list of support pairingChain part complementary pairing is positioned the specific site of nucleic acid structure. Specific chemical modification by specific combining nano particle,Polypeptide or protein. Described nano particle includes but not limited to streptavidin, gold grain, fluorophor etc. be easy to characterize andThe particle presenting. The appearance form of modifying comprises fluorescence signal and pattern etc., described pattern be letter, digital and other are regular orIrregular pattern.
Described nucleic acid structure comprises the nucleic acid structure with streptavidin modification, wherein one or several short chain DNA3 ' end extend two " T " as intervening sequence and one section of sequence that contains 15 nucleotides as auxiliary sequencel, then by oneShort chain DNA and this auxiliary sequencel that the 3 ' terminal modified length that has a biotin is 15 nucleotides match, thereby biotin is introducedIn structure, biotin subsequently can be in conjunction with streptavidin.
The described nucleic acid structure with streptavidin modification comprises the nucleic acid structure that is modified with letter " X " pattern, passes throughArticle 26, the short chain nucleotides of assigned address is incorporated into biotin in nucleic acid structure, thus in the time of AFM imaging with 26 pixelsMode present the pattern of letter " X ".
Described auxiliary sequencel is GGAAGGGATGGAGGA, and described length is that the sequence of the short chain DNA of 15 nucleotides isTCCTCCATCCCTTCC-biotin。
Interchain exchanges a synthetic method for the nucleic acid structure of being reached by support DNA, refers to and in single container, makes supportDNA mixes with multiple short chain DNA, carries out annealing reaction and form nucleic acid structure in cushioning liquid, and wherein short chain DNA's is mole denseDegree is more than or equal to 2 times of support DNA molar concentration.
Above-mentioned interchain exchanges the synthetic method of the nucleic acid structure of being reached by support DNA, comprises the following steps:
One, sample preparation
Equal-volume is drawn and is formed the required whole short chain DNA of single nucleic acid structure, and supplementary deionized water makes final mixingThe concentration of closing every short chain DNA in solution is 50 to 4000nM;
It is 50 to 500nM that support M13mp18 is mixed with to initial concentration with pure water;
Two, annealing reaction
The short chain DNA of 5 to 50nM support DNA and 10 to 500nM is blended in cushioning liquid, reaction vessel is imposed and moved backFire program.
Three, separation and purification
Annealing finish after, by reactant mixture by non-sex change agargel electrophoresis separate reacted mixture, so that targetStructure is separated physically with other structures.
The concrete steps of described separation and purification are that the reactant mixture after annealing is applied to the agar that is placed in ice-water bathGel electrophoresis, the lower target gel band of cutting subsequently, uses fine lapping rod that gel band is squeezed into fritter, (example under certain rotating speedAs 1000g) lower centrifugal several minutes, reclaim the target product being purified.
The process of described cycle of annealing is 1) or 2).
1) temperature of described cycle of annealing is reduced to 60 with arbitrary temperature of speed from 90 to 60 degree of 2-120 minute/degreeTo arbitrary temperature of 4 degree. Cycle of annealing can be lowered the temperature after 10 to 100 minutes at the high-temperature residence of 100 to 70 degree again. Be inWhen different temperatures is interval, cooling rate can be different
2) temperature of described cycle of annealing is steady temperature, and described steady temperature is the arbitrary temperature between 80 to 20 degree
The invention provides multiple nucleic acids structure and synthetic method thereof, is a kind of variant technology based on DNA paper folding art, purportUtilize support DNA to reach interchain exchange in realization and form the nucleic acid structure with controlled size, shape, complexity and modification. CoreAcid structure completes by the self assembling process of support DNA and short chain DNA, and productive rate is higher, designs easyly, and can synthesize simultaneouslyTwo-dimensional structure and the three-dimensional structure of precognition.
Brief description of the drawings:
Fig. 1 is the design diagram of a kind of two-dimentional nucleic acid structure in the present invention;
Fig. 2 is the afm image of the two-dimentional nucleic acid structure corresponding with Fig. 1;
Fig. 3 is the sketch of a kind of three-dimensional nucleic acid structure in the present invention;
Fig. 4 is the design diagram of the three-dimensional nucleic acid structure corresponding with Fig. 3;
Fig. 5 is the folding cycle path profile of the three-dimensional nucleic acid structure medium-height trestle DNA corresponding with Fig. 3;
Fig. 6 is the TEM image of the three-dimensional nucleic acid structure corresponding with Fig. 3;
Fig. 7, Fig. 8 are the design diagram that two kinds in the present invention have the two-dimentional nucleic acid structure in hole, wherein Fig. 7 representativeBe the 25 spiral nucleic acid structures of chain exchange cycle 26 of symbol " universe " pattern, Fig. 8 representative be that the chain of symbol " female " pattern is handed overChange the 25 spiral nucleic acid structures in cycle 26.
Fig. 9 is the afm image of the two-dimentional nucleic acid structure with hole corresponding with Fig. 7;
Figure 10 is the afm image of the two-dimentional nucleic acid structure with hole corresponding with Fig. 8;
Figure 11 is the design diagram of a kind of nucleic acid structure with streptavidin modification in the present invention;
Figure 12 is corresponding with Figure 11 afm image with the nucleic acid structure that streptavidin modifies;
Figure 13 is the flow chart that the annealed process of support DNA and several short chains DNA forms specific nucleic acid structure, and shouldThe afm image of structure, before annealing, support DNA exists with the form of long strand cyclic DNA.
Detailed description of the invention
The present invention improves and supplements based on one to DNA paper folding art, by support DNA but not the chain of short chain DNA hand overChange and can construct two dimension or three-dimensional nucleic acid structure simultaneously, in its broader sense, relate to and prepare the controlled size of Nano grade, shapeThe nucleic acid structure of shape, complexity and modification.
The invention provides the multiple controlled sizes of preparation, shape, pattern and the two dimension of complexity, the side of three-dimensional nucleic acid structureMethod, is prepared by the self assembling process of support DNA and short chain DNA, and this self assembling process comprises at high temperature combined supportDNA, short chain DNA and cushioning liquid also progressively reduce temperature by certain procedures, to promote sequence-specific combination. Term " group certainlyDress " refer to short chain DNA and support DNA for example, to anneal each other without the mode of external control (adding in turn short chain DNA)Process.
Nucleic acid structure provided by the invention comprises to be scheduled to carry out the short of base complementrity pairing with known way and support DNAChain DNA, therefore, the position of understanding every short chain DNA in resulting structures provides addressability for this nucleic acid structure. Correspondingly,Can the short chain DNA of assigned address be deleted or be modified and obtain the nucleic acid structure with specific hole and pattern.
1, two-dimentional nucleic acid structure:
Two dimension nucleic acid structure is made up of in the mode of specific binding each other support DNA and short chain DNA, is forming nucleic acid knotBefore the annealing process of structure, support DNA exists with the form of ring-type long-chain DNA, and every short chain DNA is less than 400 to containThe single stranded form of nucleotides exists.
Support DNA in nucleic acid structure and short chain DNA form double-helical mode by the pairing of base complementrity each other and exist,These double helixs also can be called as spiral in the text of equal valuely.
It is the design diagram of 25 spiral rectangle nucleic acid structures of 26 nucleotides that Fig. 1 provides chain exchange cycle. WeDefinition, from top to bottom, the position of every row spiral is 1,2,3 ... 26, in every row spiral, from left to right, each nucleotidesPosition is 1,2,3 ... until end. Usually, the 0th spiral of support DNA will not mate with short chain DNA, and with long singleChain form exists, and since the 26th, 27 nucleotides of the 1st spiral, every 52 nucleotides, a support DNA chain exchange is setSite is to (crossover), left several the 26th, 27,78,79 ..., until spiral finishes. The 2nd spiral is to the 2nd row spiral shell reciprocalThe 26th, 27 nucleotides that revolve start, and every 26 bases, chain exchange site pair are set, and 26,27,52,53 ..., straightFinish to spiral. The 52nd, 53 nucleotides of last column spiral start, and every 52 nucleotides, an one/chain exchange position are setPoint is right. Exchange site to locating at each chain. Each nucleotides all with adjacent a line spiral (lastrow or next line, replace successively)The nucleotides of same position connects. Every a line spiral is arranged parallel to each other and the few nucleotide that comprises can be identical. Spiral number andThe few nucleotide that every row spiral comprises depends on designer's demand, and the total nucleotide number that the nucleic acid structure of design comprises should be less thanThe few nucleotide of support DNA. Fig. 2 is the afm image of this nucleic acid structure, and its shape conforms to design with size.
In fact, for avoiding support DNA issuable distorting stress (twist) accumulation in folding process, to eachRow spiral, from first nucleotides, makes the cycle in chain exchange site increase a nucleotides every 104 nucleotides, withJust meet the multiple (each spiralization cycle is 10.5 nucleotides) of DNA spiralization cycle, eliminate possible impact. Therefore, withThe 3rd spiral is example, and actual chain exchange right position, site should be 26-27,52-53,78-79,105-106 is by that analogy, straightFinish to spiral.
For the short chain DNA in this two dimension nucleic acid structure, its arrangement position is with reference to following regular: for the 0th row spiral, noAdd short chain DNA to mate. Walk to footline spiral for the 1st, from the 14th nucleotides, place successively adjacent (centreTight) length be that the short chain DNA of 26 nucleotides mates and forms double helix, until the position of the 14th nucleotides reciprocalPut. All short chain DNA all exist with parallel mode in helical structure. Therefore for the every a line spiral except the 0th row, from beginning to endTo respectively have 13 nucleotides not form double helix, the object of this measure is to avoid nucleic acid structure in unnecessary the gathering of border generation each otherCollection.
It is 26 11 spiral nucleic acid structures that two dimension nucleic acid structure also can design chaining exchange cycle, and chain exchange cycle is 1623 spiral nucleic acid structures etc. For the two-dimentional nucleic acid structure of other shape the present invention relates to, size, all can be with reference to above-mentioned sideMethod designs, and total spiral number that nucleic acid structure comprises can be 2 to 200 row, and the nucleotides that every row spiral comprises can be 60To 6000. The length of every short chain DNA is less than 400 nucleotides. Chain exchange cycle can be between 4 to 416.
2, three-dimensional nucleic acid structure
Three-dimensional nucleic acid structure is the interchain exchange based on support DNA equally, carries out base by support DNA and short chain DNAThe mode of complementary pairing forms.
It is the sketch of 35 spiral three-dimensional nucleic acid structures of 26 nucleotides that Fig. 3 provides chain exchange cycle, every row spiralNumber as shown in the figure, the double helix that is of five storeys altogether, every layer of double helix overlaps completely in the projection of the 1st helical layer plane of living in. Every layer of spiral shellRevolve by 7 row double helixs and form, the nucleotides that every row double helix is all arranged and contained same number with parallel mode. Therefore, this threeDimension nucleic acid structure presents cubical shape in space.
The design diagram of corresponding three-dimensional nucleic acid structure as shown in Figure 4. We define 5 ' of the folding starting point of support DNAIn first nucleotides place of the first row spiral, to the 1st row spiral, from the 26th, 27 nucleotides, establish every 52 nucleotidesPut chain exchange site pair, the 26th, 27,78,79 ... individual spiral place, until spiral finishes. To last column spiral (35 row spirals), since the 52nd, 53 nucleotides, every 52 nucleotides, a chain exchange site pair is set, until spiral knotBundle. To remaining every row double helix, from the 26th, 27 nucleotides, every 26 nucleotides, a chain exchange site pair is set,I.e. the 26th, 27,52,53,78,79 spiral places, until spiral finishes. Every a line spiral exchanges site and adjacent lines spiral shell by chainPrecession row connects, and this is adjacent refers to adjacent at schematic diagram display position, for example the 2nd row spiral and the 1st row, the 3rd row, the 13rdRow spiral is adjacent.
The folding path of support DNA:
Support DNA has completed 4 identical folding cycles in this three-dimensional nucleic acid structure, and each cycle is different by two kindsFolding path composition. Taking first folding cycle as example, as shown in Figure 5. From 5 ' end, support DNA is reaching the 1st row spiralThe 1st place chain exchange site to rear, enter the 2nd row spiral, be back to subsequently the 1st nucleotides place of the 2nd row spiral, form oneIndividual " U " shape loop, enter subsequently the 3rd row spiral, and enter the 4th row spiral in first chain exchange site of the 3rd row spiral.In this way, support DNA, according to natural sequence, covers the 1st, 2,3 successively ... 35 row spirals.
Support DNA reaches after the 35th row spiral, enters the 22nd row spiral, and be back in the 1st chain exchange site to locatingFirst chain exchange site pair of the 22nd row spiral, enters the 21st row spiral subsequently. In first chain exchange of the 21st row spiralSite, to locating to enter eighth row spiral, exchanges site to locating to enter the 7th row spiral at the 2nd chain of eighth row spiral. In this way,Support DNA, according to the arrangement of vertical direction spiral in schematic diagram, covers the the the the 35th, 22,21,8,7,6,9 successively ... then get back toThe 1st row spiral.
Remaining folding cycle all according to said method carries out, it should be noted that support DNA the chain exchange site pair of processAccording to the corresponding change of folding periodicity. For example, in the 2nd folding cycle, the chain exchange site of support DNA process is to being every rowThe 2nd and the 3rd, support DNA completes after all folding cycles, returns to last nucleotides place of the 1st row spiral,Between 1 row spiral first and last nucleotides, be connected by long single stranded DNA, to meet the condition of support DNA as cyclic DNA.
Short chain DNA in the three-dimensional nucleic acid structure that is 26 for chain exchange cycle, its arrangement position is with reference to following regular: rightIn the 1st to footline spiral, from the 14th nucleotides, the length of placing successively adjacent (middle tight) is 26 nucleotidesShort chain DNA mate and form double helix, until the position of the 14th nucleotides reciprocal. All short chain DNA are at helical structureIn all exist with parallel mode. To every a line spiral, head and the tail will respectively have 13 nucleotides not form double helix, so that avoid may beThe accumulation phenomena that border produces.
As shown in Figure 6, its physical dimension conforms to design the TEM image of above-mentioned three-dimensional nucleic acid structure.
For the three-dimensional nucleic acid structure of other shape the present invention relates to, size, all can establish with reference to said methodMeter, three-dimensional nucleic acid structure can comprise 2 to 100 layers of double helix, and the spiral number that every helical layer comprises can be 2 to 100 row, every rowSpiral can comprise 60 to 3000 nucleotides. The length of every short chain DNA is less than 400 nucleotides. Chain exchange cycle canBetween 4 to 416 like this. The final three-dimensional nucleic acid structure forming is rendered as the shape of cuboid in space.
Above design process and final support DNA sequence dna are inputted, the output of short chain DNA sequence dna can be in Cadnano softwareComplete. Certainly, final, we can design the nucleic acid structure with length-specific and width, and accurate in solution structureThe position of every short chain DNA and sequence. Whereby, can be by selectively deleting that short chain DNA or the short chain DNA to ad-hoc location enterRow is modified, and obtains controllable shape, size and has hole or the two and three dimensions figure of pattern.
3, there is the nucleic acid structure in hole
Position based on to every short chain nucleotides in nucleic acid structure, the accurate understanding of length, can specify by eliminatingOne or more short chain nucleotides in nucleic acid structure, make support DNA in this position not participation structure form to demonstrate hole.Then adopt identical annealing process, finally obtain thering is controllable shape, the nucleic acid structure in the hole of size.
Consider the short chain nucleotides that can directly get rid of optional position in nucleic acid structure, the hole side of comprising formingShape, polygon, cross and other rule or irregular shape. Because the precision of AFM probe is limited, design holeLength and width should be to facilitate the measurement of AFM more than 10nm. Therefore the short chain DNA, being left out also has certain quantity. Real at someIn example, can be by deleting specific 20 short chain nucleotides to form criss-cross hole, by deleting respectively ad-hoc location60 and 48 nucleotides form the hole of representative " universe " and " female " implications. Fig. 7 and Fig. 8 have shown respectively with in Eight DiagramsThe design diagram of 25 spiral nucleic acid structures of the chain exchange cycle 26 in the hole of " universe " hexagram pattern and " female " hexagram pattern. In designIn figure, the short chain DNA of assigned address is directly left out and is not participated in annealing process. Left out rear formation by short chain DNAHole embody the pattern of design. The afm image of the nucleic acid structure in respectively corresponding " universe " and " female " hole of Fig. 9 and Figure 10, itsHole occurs that position conforms to design with size, shape, proves can obtain the nucleic acid knot with specific hole by the methodStructure.
Nucleic acid structure in the present invention has also considered to form by the folding path of amendment support DNA the method in hole. ?Interchain exchange is carried out in the position that frame DNA exchanges 13 nucleotides in site at 8 adjacent chains of distance, and (substituting original distance is 26The chain exchange site of individual nucleotides), therefore forming a length is 26 nucleotides, width is the region, hole of 4 spirals. ExamineConsider the distorting stress that may cause to nucleotide deletion, between the corresponding site in these 8 chain exchange sites, increase by four nucleosidesAcid is as intervening sequence, to eliminate the effects of the act. Can form the nucleic acid knot with hole of specific size, shape by the methodStructure. And because support DNA no longer exists in region, hole, the hole of observing under AFM will approach theoretical size more.
Two-dimentional nucleic acid structure in the present invention can also form hole by direct increase intervening sequence. Support DNA'sThe chain exchange site of four same positions is deleted, is 4 nucleotides and increase altogether 4 length in adjacent chain exchange siteIntervening sequence, forming a length is 52 nucleotides, width is the region, hole of 4 spirals. The method equally can shapeBecome to have the nucleic acid structure with hole of specific size, shape.
4, there is the nucleic acid structure of modification
Nucleic acid structure in the present invention can carry out chemical modification, tool at single specific site in multiple range of structuresHave the nucleic acid of chemical modification to can be used as short chain DNA and be directly positioned specific site, or by with short chain DNA not with supportThe strand part complementary pairing of DNA pairing is positioned the specific site of nucleic acid structure indirectly. Specific chemical modification is by specialCombining nano particle, polypeptide or the protein of property. Described nano particle includes but not limited to streptavidin, gold grain, glimmeringLight group etc. is easy to the particle that characterizes and present. The appearance form of modifying comprises fluorescence signal and pattern etc., and described pattern is wordMother, numeral and other regular or irregular patterns.
The combination of streptavidin and biotin is the current known the highest noncovalent interaction of intensity, and is modified with biologyThe short chain DNA of element is commercially produced. The accurate understanding of every short chain nucleotide position in nucleic acid structure is provided canAddressing. Therefore, the short chain nucleotides of ad-hoc location in structure can be extended to one group of sequence, then by biotin modification with thisOn the short chain nucleotides that prolongation sequence matches, then by the specific binding of streptavidin and biotin, under AFM, seeMeasure the nucleic acid structure that is modified with predetermined pattern. Consider and can repair the short chain DNA of optional position known array, lengthDecorations, the method can realize and obtain the nucleic acid structure that is modified with any reservation shape.
In some instances, for all sites that need biotin modification in nucleic acid structure, the short chain of its present positionNucleotides will extend two " T " as intervening sequence and one section of sequence that contains 15 nucleotides at 3 ' end(GGAAGGGATGGAGGA), as auxiliary sequencel, the short chain nucleotides of 3 ' end biotin modification contains 15 nucleotides, itsSequence is TCCTCCATCCCTTCC-biotin, matches with this auxiliary sequencel. Before annealing process, by support DNA, short chainDNA, the short chain DNA that contains biotin modification mix. Before AFM scanning, the streptavidin of 1uM is joined carrying outIn testing sample, leave standstill 10 minutes and wait for streptavidin and the abundant combination of biotin, can observe and be modified with specific patternNucleic acid structure.
Figure 11 shown based on the method, and being modified with letter " X " chain exchange cycle is 25 spiral nucleic acid of 26 nucleotidesThe design drawing of structure. As shown in the figure, letter " X " contains 26 decorating sites, and the short chain DNA at decorating site place is increased to two" T ", as intervening sequence and 15 nucleotides as auxiliary sequencel, a segment length is that 15 nucleotides 3 ' is terminal modified a biotinShort chain DNA and this auxiliary sequencel match and participate in annealing process, are finally attached in the nucleic acid structure of preparation, make chainEnzyme Avidin can be combined with biotin in fixed position. Figure 12 is and the afm image of nucleic acid structure corresponding to letter " X ". Its chainEnzyme Avidin binding site conforms to design with final display pattern, proves can realize obtaining by the method to have specific repairingThe nucleic acid structure of decorations.
For the modification before annealing process, its prerequisite should ensure that this short chain DNA additionally adding can not affect the shape of structureBecome. The present invention has also considered to modify as alternative after annealing process. For example, after completing, annealing will be modified with againThe short chain DNA of biotin joins in sample, then under 37 °, cultivates 10 minutes. Similarly, before AFM scanning, add chain enzymeThen Avidin is observed.
5, support DNA
The support DNA adopting in the present invention is M13mp18, and M13mp18 is a ring-type in m13 phage genome DNASingle strand dna, contains 7249 nucleotides altogether, obtains and commercially produces.
6, short chain DNA
The design of amplifying nucleic acid structure of the present invention and preparation process relate to hundreds of different short chain DNA, and short chain DNA can lead toCross its length, sequence characterizes, and by it, the sequence of the position in structure and support DNA determines, follows base complementrity and joinsTo principle. For single nucleic acid structure, its every short chain DNA comprising is unique, only in structure, occurs once; For notThe structure of same nucleic acid, short chain DNA can be identical.
Short chain DNA can synthesize in vitro, and the method for the synthetic short chain DNA of automation is known and in this areaCommercial operation.
The invention provides multiple nucleic acid structures, these multiple structures can have homogeney in various degree, expect multipleIn nucleic acid structure, having the structure of certain percentage mutually the same with regard to size, shape, complexity, can be also to have a certain spyIn the structure of levying at least 50%, 60%, the 70%th, homogeneity. In some cases, its member of multiple nucleic acid structures can be baseMonodispersed in basis, substantially singly disperse to refer at least 50%, 60%, 70% and there is approximately identical size, shape, complexityDegree.
The size of nucleic acid structure can by its two dimension or three-dimensional dimension represent, this dimension can be even sub-micron of nanometerThe size of rank. In some instances, two-dimentional nucleic acid structure is that about 200nm takes advantage of 40nm; Three-dimensional nucleic acid structure is that about 62nm takes advantage of26nm takes advantage of 19nm. The size of nucleic acid structure can also be rendered as the nucleotides that its double helix number comprising and every a line spiral containNumber. For example a certain two-dimensional structure in the present invention contains 11 row double helixs, and every row double helix comprises 1154 nucleotides.
The present invention is by combined support DNA and short chain DNA in single annealing reaction, to obtain required size, shape, multipleAssorted degree and the nucleic acid structure of modifying. In some embodiments, can implement high temperature to reaction vessel, Slow cooling carries out subsequentlyAnnealing, high temperature can be approximately 90 degree, 85 degree, the temperature of 80 degree, cooling procedure can be cooled to room temperature by solution, and 10 DEG C, 4 DEG C are veryTo lower. Cooling cycle can be 10 hours, and 15 hours, 25 hours even more of a specified duration. In other examples, can be to reaction vesselImplement constant temperature processing, constant temperature can be approximately 65 DEG C, and 60 DEG C, 55 DEG C, the constant temperature phase can be 10 hours, and 15 hours, 25 hours evenMore of a specified duration.
The homogeney degree of multiple nucleic acid structures can be used many technology to measure, and includes but not limited to AFM, TEMAnd gel electrophoresis. Described in example, these technology are measured for the homogeney to gained nucleic acid structure.
At Fig. 1, Fig. 4, Fig. 7, Fig. 8, Tu11Zhong, short chain DNA starts, adds respectively the mode of numeral number below with a, d, eIndicate, as a1.1. The sequence of corresponding short chain DNA is listed in sequence table. The sequence of for example a1.1 is as shown in SEQIDNO3.
7, synthetic method
The invention provides by the method for annealing process nucleic acid structure, by after support DNA and short chain DNA mixing,Can be at single container but be not limited to mix in pipe, hole, bottle. Solution generally cushions, but annealing reaction also canIn the situation that there is no cushioning liquid, carry out. Cushioning liquid can comprise bivalent cation but be not limited to Mg2+. Cation concn is logicalBe often 5 to 20mM, but also can change. Solution comprises EDTA or other enzyme inhibitor conventionally, to stop the degraded of nucleotides. ?Annealing mixed solution in, the concentration of short chain DNA depends on the concentration of support DNA, be generally support DNA molar concentration 5 to20 times, support DNA can use the molar concentration of 10nM. The volume of whole annealing mixed liquor can be 20ul, 30ul or moreHigh.
Annealing reaction is by carrying out solution heating Slow cooling. Heating-up temperature should be enough high, guarantees short chain DNA notCan carry out combination with incomplementarity sequence, and for example hairpin structure of any undesirable secondary structure is unwind. Can pass through willReaction vessel is placed in hot bath, PCR (PCR device) or other attemperating units and heats. Container can be at heightTemperature is lower cultivates several seconds or several minutes. In general, the cultivation of 1 to 20 minute is enough.
After cultivation at high temperature completes, temperature can decline in many ways, can use the method for automation control,Maintain a period of time in a certain temperature, once declining subsequently or spending more, continue to maintain a period of time, carry out in this way alwaysUntil the minimum temperature that control program is set.
Can implement high temperature to reaction vessel, Slow cooling is annealed subsequently, and high temperature can be approximately 90 degree left and right, coolingIt is even lower that process can be cooled to solution room temperature. Cooling cycle can be 10 hours, and 15 hours, 25 hours even more of a specified duration.
Annealing reaction also can be carried out by solution is carried out to constant temperature processing, thermostat temperature conventionally 65 DEG C to 55 DEG C itBetween, constant temperature time is conventionally more than 10 hours.
Embodiment has described a concrete annealing process. Many the short chain DNA of the support DNA of 10nM and 200nM are mixedBe combined in Tris-EDTA/10mMMg2+In cushioning liquid, solution is heated to 90 DEG C, the annealing of passing through subsequently approximately 26 hours is slowBe cooled to 4 DEG C.
An other set condition that can be used for annealing process also comprises Te/Mg2+Buffer solution (10mMTris, PH8.0,2mMEDTA、15mMMg2+) and at 60 DEG C constant temperature 24 hours.
Annealing process is without the strict concentration of controlling short chain DNA and support DNA, also without strictly observing Chemical Measurement.
After annealing finishes, reactant mixture can directly be analyzed or further separate. Can pass through the non-change of 1-2%Property agargel electrophoresis separate reacted mixture, so that object construction separates physically with other structures. Conventionally we can seeExamine single remarkable band, band can be by extraction in gel further pure via centrifugal removal short chain DNA and other impurityChange, the product after purifying can pass through AFM or TEM imaging, and this type of imaging can be reacted the size and dimension of target product, discloses pre-Know the formation of structure. Purified product also can again be carried out gel electrophoresis and observe the single band of expection.
The efficiency of annealing process can be measured by the percentage of the structure of all in the AFM visual field " fully forming ".If nucleic acid structure does not have diameter to be greater than the defect of 10nm, it is regarded as " fully forming ". According to above-mentioned standard, the present inventionIn all nucleic acid structures all have the AFM " productive rate " of more than 80% " fully form ". consider that sample is being deposited on mica tableThe infringement that face or AFM probe may stand when structure is scanned, this may be a numeral lower than actual conditions.
8, Cadnano software operation flow process:
The flow process of drawing the nucleic acid structure in the present invention by Cadnano is as follows:
8.1 Graphics Design
In the first half of software, select as required spiral number. In the latter half, the corresponding a line of the double grid of every a lineSpiral, represents two dimension or three-dimensional structure in this way, and the first half is integrally-built side view. The latter half is for overlookingFigure. Determining after spiral number, can directly on top view, draw. According to designing requirement, according to 5 ' to 3 ' foldingDirection, after the folding path of drawn support DNA, then fills the short chain DNA mating with ad-hoc location, unnecessary core successivelyThuja acid is placed in the loop (loop) of the first row spiral, completes structural design.
8.2 sequence inputs and output
Select certain nucleotides in support DNA as the starting point of 5 ' direction, to input the sequence of known support DNA,Can obtain the particular sequence of each short chain DNA. Software can generate the EXCEL formatted file that contains all short chain DNA sequence dnas.
9, specific implementation method
Embodiment 1:
As shown in figure 13, the preparation process the present invention relates to comprises following steps:
1, sample preparation:
Short chain DNA is synthesized by Bioneer company, and equal-volume is drawn and formed the required whole short chain DNA of single nucleic acid structure,It is 500nM that supplementary DEPC water makes the concentration of every short chain DNA in final mixed solution. Extension rate depends on manufacturer's ruleInitial concentration in lattice list and do not carry out extra inside and proofread and correct, therefore the Chemical Measurement of short chain DNA is without accurate control.
M13mp18 in support DNA purchases from NEB company, and it adds appropriate pure water to make initial support DNA concentration be100nM。
2, annealing reaction
The short chain DNA of 10nM support DNA and 200nM is blended in to cushioning liquid (10mMTris, pH8.0,2mMEDTA、10mMMg2+) in, impose cycle of annealing and make reaction vessel slowly cool to 4 DEG C from 90 DEG C. Temperature changing process is: 90DEG C stop 10 minutes, between 90 DEG C to 60 DEG C with 5min/ DEG C of decline, between 60 DEG C to 4 DEG C with 25min/ DEG C of decline. To move backSample application after fire in be placed in ice-water bath 2% agargel electrophoresis (use Gelred dyeing, buffer solution is 0.5 ×TBE/10mMMgCl2). The lower target gel band of cutting subsequently, uses fine lapping rod that gel band is squeezed into fritter, under 1000gCentrifugal 3 minutes, reclaim the target product being purified.
3, AFM imaging
Mixed solution after annealing can directly carry out AFM imaging, measures after also can using gel electrophoresis purifying again.The AFM Image-forming instrument that the present invention adopts is the multimode8 of Bruke company. By the sample after annealing or gel electrophoresis purifyingDilute 5 times, get 2ul dropping and tear flat mica surface just, add subsequently 50ul1 × TE/10mMMg2+, to promote nucleic acid structureSedimentation, also can according to actual conditions change sample dilution ratio or drip volume close in the visual field to meet nucleic acid structureThe suitable ratio of arranging (in the visual field, most nucleic acid structures can be known identification, without packing phenomenon). Use with SNL-10 silicon nitrideThe AFM of cantilever scans under liquid phase intelligent scanning pattern (ScanAsystinFluid). Conventionally choose 1um's to 10umSweep limits. When scanning, the value of instrument setpoint is fixed on between 0.02-0.03, contact force is excessive to be entered structure to avoidRow infringement, other parameter can be regulated voluntarily by instrument.
4, TEM imaging
For three-dimensional nucleic acid structure, we observe the three-dimensional appearance of product conventionally with TEM. By 3.5ul testing sample(1-5nM) be added drop-wise on glow discharge coating TEM grid and leave standstill 4 minutes, use subsequently containing 2% formic acid of 25mMNaOH twoOxygen uranium aqueous solution dyeing 5 seconds operates FEITecnaiG2F20TWIN field transmission electron microscope and becomes under 80kVPicture.
Embodiment 2
Streptavidin mark
By the short chain DNA of 10nM support DNA, 100nM, (the short chain DNA of ad-hoc location terminal modifiedly has an auxiliary sequencel 3 'GGAAGGGATGGAGGA also extends TT as intervening sequence), the complementary short chain DNA of 40uM3 ' biotin modification(TCCTCCATCCCTTCC) be blended in cushioning liquid (10mMTris, pH8.0,2mMEDTA, 10mMMg2+) in, impose and move backFire program makes reaction vessel slowly cool to 4 DEG C from 90 DEG C. Temperature changing process is: 90 DEG C stop 10 minutes, at 90 DEG C to 60Between DEG C with 5min/ DEG C of decline, between 60 DEG C to 4 DEG C with 25min/ DEG C of decline. Before AFM imaging, will anneal or gel electricity5 times of Sample Dilutions after swimming purifying, get 2ul dropping and tear flat mica surface just, add 50ul1 × TE/10mMMg2+, withAfter add the streptavidin solution of 2ul concentration 1uM, leave standstill 10 minutes, then carry out AFM scanning.
10, measurement and statistics
The NanoscopeAnalysis software that uses Bruke company to provide carries out measurement and the analysis of afm image, forTwo-dimensional (length of nucleic acid structure and width), should be used cross-section function, selects the structure of 20 " fully forming " to obtainObtain mean value. Use the TEM image of the three-dimensional nucleic acid structure of ImageJ software analysis of NIH, collect equally 20 sample spot and be used forThe measurement task of nucleic acid structure.

Claims (13)

1. interchain exchanges a nucleic acid structure of being reached by support DNA, it is characterized in that: be to reach interchain by support DNA to hand overTwo dimension or the three-dimensional nucleic acid structure with controlled size and shape that change formation, this nucleic acid structure comprises that one group exchanges by interchainThe parallel double spiral coupling together and one section of single stranded DNA, described nucleic acid structure by support DNA by folding, interchain exchange and withMany short chain DNA carries out specificity coupling, forms via annealing reaction, and the few nucleotide that every row double helix comprises can phaseWith, single stranded DNA is in support DNA, not form double-helical part.
2. interchain according to claim 1 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described supportDNA comprises the single stranded DNA of M13mp18 or other specified sequence; M13mp18 is a ring-type list in m13 phage genome DNASsdna molecule, contains 7249 nucleotides altogether, and its sequence is as shown in SEQIDNO1.
3. interchain exchange according to claim 1, is characterized in that: keep at a certain distance away at the each double helix of nucleic acid structurePlace arranges a pair of chain exchange site, and the folding path of support DNA enters adjacent via each double-helical chain exchange siteThe chain exchange site of double helix relevant position, thisly utilizes that support DNA is will be multiple parallel spiral exchanges site to connected by chainThe behavior connecing is interchain exchange; The double-helical chain exchange of every row site is 2 to 500 pairs, to same a line double helix, adjacent chainDistance between exchange site pair is that chain exchange cycle is 4 to 416 nucleotides.
4. interchain according to claim 1 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described nucleic acidStructure is two-dimentional nucleic acid structure, and this two dimension nucleic acid structure comprises 2 to 200 row double helixs, segment length's single stranded DNA and 20 to 1000The short chain DNA mating with support DNA at least partly; Every row double helix comprises 60 to 6000 nucleotides, chain exchange cycle be 4 to416; The length of every short chain DNA can equate and all be less than 400 nucleotides to same structure. For single nucleic acid structure,Its every short chain DNA comprising is unique, only in structure, occurs once; For different nucleic acid structures, short chain DNA canIdentical.
5. interchain according to claim 4 exchanges the two-dimentional nucleic acid structure of being reached by support DNA, it is characterized in that: described inTwo dimension nucleic acid structure is square, polygon or other rule or irregular two dimensions forming in the length restriction of support DNAFigure; The difformity of two dimension nucleic acid structure is reached by changing the folding path of support DNA and the matched position of short chain DNA.
6. interchain according to claim 1 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described nucleic acidStructure is the 3-D solid structure of multilayer nucleic acid composition, comprises segment length's single stranded DNA and 20 to 1000 at least part of and supportsThe short chain DNA of DNA coupling, 2 to 100 layers of double helix, the spiral number that every helical layer comprises is 2 to 100 row, every row spiral comprises 60To 3000 nucleotides, chain exchange cycle is 4 to 416, and the length of every short chain DNA can equate and all be less than same structure200 nucleotides; For single nucleic acid structure, its every short chain DNA comprising is unique, only in structure, occurs once;For different nucleic acid structures, short chain DNA can be identical.
7. interchain according to claim 6 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described three-dimensionalNucleic acid structure is cube, polyhedron or other rules forming in the length restriction of support DNA or irregular three-dimensional knotStructure; The difformity of three-dimensional nucleic acid structure is reached by changing the folding path of support DNA and the matched position of short chain DNA.
8. interchain according to claim 1 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described nucleic acidStructure is the nucleic acid structure with hole, and this hole, by getting rid of one or more short chain DNA in nucleic acid structure, makes supportDNA in this position not participation structure form and reach, that described hole comprises is square, polygon and other rule or irregular shapeShape.
9. interchain according to claim 1 exchanges the nucleic acid structure of being reached by support DNA, it is characterized in that: described nucleic acidStructure is the nucleic acid structure at single specific site in multiple range of structures with chemical modification, has the core of chemical modificationAcid directly as short chain DNA or indirectly by with not the determining with the strand part complementary pairing of support pairing of short chain DNABe positioned at the specific site of nucleic acid structure; Described chemical modification is by specific combining nano particle, polypeptide or protein; DescribedNano particle comprises streptavidin, gold grain or fluorophor; The appearance form of modifying comprises fluorescence signal and pattern, described inPattern comprises letter, numeral and other regular or irregular patterns.
10. interchain exchanges the synthetic method of the nucleic acid structure of being reached by support DNA, it is characterized in that, makes to prop up in single containerFrame DNA mixes with many short chain DNA, in cushioning liquid, carry out annealing reaction form nucleic acid structure, wherein short chain DNA moleConcentration is more than or equal to 2 times of support DNA molar concentration.
11. interchains as claimed in claim 10 exchange the synthetic method of the nucleic acid structure of being reached by support DNA, and its feature existsIn, comprise the following steps:
One, sample preparation
Equal-volume is drawn and is formed the required whole short chain DNA of single nucleic acid structure, and supplementary deionized water makes final mixing moltenIn liquid, the concentration of every short chain DNA is 50 to 4000nM;
It is 50 to 500nM that support M13mp18 is mixed with to initial concentration with pure water;
Two, annealing reaction
The short chain DNA of 5 to 50nM support DNA and 10 to 500nM is blended in cushioning liquid, reaction vessel is imposed to annealing journeyOrder;
Three, separation and purification
After annealing finishes, reactant mixture is separated by non-sex change agargel electrophoresis, so that object construction and other structuresSeparate physically.
12. synthetic methods as claimed in claim 11, is characterized in that: the concrete steps of described separation and purification are to annealAfter reactant mixture be applied to the agargel electrophoresis that is placed in ice-water bath, the lower target gel band of cutting, uses and carefully grinds subsequentlyGel band is squeezed into fritter by frotton, centrifugal several minutes, reclaims the target product being purified.
13. synthetic methods as claimed in claim 11, is characterized in that: the time of described cycle of annealing be 2 minutes to 200 littleTime, the temperature-controlled process of cycle of annealing comprises following two kinds:
1) temperature of described cycle of annealing is reduced to 60-4 degree with the speed of 2-120 minute/degree from 90-60 degree, or 100 to 70The high-temperature residence of degree is lowered the temperature after 10 to 100 minutes again; For different temperature ranges, cooling rate can be different;
2) temperature of described cycle of annealing is steady temperature, and described steady temperature is the arbitrary temperature between 80 to 20 degree.
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