CN109389225A - Molecular Logic Gates and its construction method based on DNA nanotripods regulation fluorescent small molecule and graphene oxide interaction - Google Patents
Molecular Logic Gates and its construction method based on DNA nanotripods regulation fluorescent small molecule and graphene oxide interaction Download PDFInfo
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Abstract
The invention discloses a kind of Molecular Logic Gates and its construction method to be interacted based on DNA nanotripods regulation fluorescent small molecule and graphene oxide.In the DNA nanotripods of self assembly, a center chain forms Center-triangle by three side chain partial hybridizations, and the remainder of three side chains extends to form single stranded DNA leg as signal reception chain, and center chain loads fluorophor ROX.Signal reception chain is adsorbed in surface of graphene oxide when hybridize with signal input chain, signal reception chain with signal input chain hybridize become double-strand after from surface of graphene oxide desorption.The present invention inputs the compatibility that chain adjusts DNA nanotripods and graphene oxide by combination input unlike signal, keep DNA nanotripods different in the configuration states of surface of graphene oxide, to regulate and control the interaction of fluorophor ROX and graphene oxide that it is loaded, different fluorescence signals is generated, the building of different kinds of molecules logic gate is realized.
Description
Technical field
The invention belongs to molecular computer technical fields, and in particular to one kind is small based on DNA nanotripods regulation fluorescence
The Molecular Logic Gates and its construction method of molecule and graphene oxide interaction.
Background technique
In hyundai electronics computer, electronic logic door is the basic module of integrated circuit.Molecular Logic Gates are patrolled with electronics
Collecting door has similitude, and by suitably designing, boolean calculation rule is similarly applied to molecule reaction system, realizes defeated
Enter the conversion of signal to output signal.The input signal of chemistry, biology and physics can be transformed into certain by Molecular Logic Gates
Output signal, therefore, Molecular Logic Gates are the bases for constructing molecular computer.Molecular logic operation system is constructed, chemistry is utilized
Or biochemical reaction Analogical Electronics system carries out logical operation, realizes information processing on a molecular scale, is computer section
It learns, chemistry and biology combines and the emerging research field that grows up.In the present art, transported using molecular logic
The smart molecule device for calculating system construction nanoscale illustrates biological phenomena for chemistry, bio-sensing, and medical diagnosis is treated,
The intelligent control of even bioprocess has become possible.
In recent years, people have developed a large amount of structure-controllables, addressable by position DNA from group using DNA nanotechnology
Fill nanostructure.The interaction of DNA and natural or synthetic molecule is controllable, can design, and therefore, nanostructure is in three-dimensional arrangement function
Can group, have as die casting and be arbitrarily designated the inorganic nano material of three-dimensional structure, encapsulation and release drug, protein and
Nano material, and assign new property to the guest molecule of encapsulation etc. and all show great potential.Recently, Yi Xieyan
Study carefully and report that compact-sized Self-assembled DNA nanostructure is easy to by cellular uptake, moreover, single-stranded compared to simple DNA and double
Chain, DNA nanostructure are more stable in physiological conditions.These peculiar properties are to use DNA nanostructure as carrier, will be a variety of
Function element is loaded into cell according to a certain percentage, and provides the foundation in the function intracellular of being performed in unison with them.Therefore, DNA
Nanostructure introduces DNA logic circuit and will can analyze a variety of measurement signals in vivo for design and make the intelligence suitably responded
System provides general platform, such as DNA nano-carrier, can execute targeting in a manner of a kind of stimuli responsive type as required
The release of conveying task and intelligent control tote.Therefore, the building that DNA nanostructure is used for molecular logic circuit is caused
Extensive research interest.
Existing DNA logic gate system, most of is based on simple DNA nanostructure, such as DNA double-strand, hair clip knot
The buildings such as structure, DNA intersection construction.Such design method causes function element to lack versatility, so that the logic constructed
Device architecture dispersion, circuit is lengthy and jumbled, hinders basic logical gate and is cascaded into multistage complex logic circuit, increases construction logic door
Cost.Using Integrated design strategy, multiple function element can be assembled into one, can be increased using Self-assembled DNA nanostructure
Its strong versatility constructs a variety of Different Logic doors, greatly simplifies the design and operation of Molecular Logic Gates.
Summary of the invention
The object of the present invention is to using DNA self-assembled nanometer technology by multiple function element be assembled into one and with oxidation
Graphene combines, and constructs the design that a universal molecule platform is used for different kinds of molecules logic gate.
To solve its technical problem, received the technical solution adopted by the present invention is that assembling a DNA using DNA nanotechnology
Rice tripod (DNA nanotripod), and regulate and control on nanoscale as bracket the fluorophor ROX of its load with
The interaction of graphene oxide, as shown in Figure 1, realizing the building of different kinds of molecules logic gate.It specifically includes:
One, a kind of molecular logic based on DNA nanotripods regulation fluorescent small molecule and graphene oxide interaction
Door:
Including graphene oxide, DNA nanotripods (DNA nanotripod) and multiple single-stranded DNA sequences, DNA nanometers
Tripod includes a center chain and three side chains, and a center chain passes through in a part hybridization formation therein of three side chains
Heart triangle, the remainder of three side chains extend out to form single stranded DNA leg from center Atria vertex respectively, utilize
DNA is end modified to make center chain load fluorophor ROX, using the single stranded DNA leg of nanotripods as signal reception chain, knows
It is not multiple as signal input chain single-stranded DNA sequences, multiple single-stranded DNA sequences as signal input chain respectively with three signals
Reception chain Complementary hybridization；Signal reception chain is adsorbed in surface of graphene oxide when not hybridizing with signal input chain, and signal receives
Chain hybridize with signal input chain become double-strand after from surface of graphene oxide desorption.
The present invention is combined input by the way that homotactic single stranded DNA is inputted chain as signal, with DNA nanometer three
Foot prop is as bracket, the interaction of regulating DNA nanotripods and graphene oxide, and then the regulating DNA on nanoscale
The interaction of the fluorophor ROX and graphene oxide of nanotripods load, as shown in Figure 1, building forms different kinds of molecules
Logic gate.
Center chain, single stranded DNA leg and the signal input chain is single stranded DNA.Three single stranded DNA legs and center chain
Between, single stranded DNA leg and signal input chain between by base pair complementarity hybridization combination.5 ' ends of the center chain connect
It is connected to fluorophor ROX.
The base sequence of the center chain of the DNA nanotripods is SEQ ID NO.1, the alkali of three signal reception chains
Basic sequence is respectively SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.
The DNA nanotripods package assembly is as follows:
Multiple signals input chain I1, I2, I3, I1 ', and base sequence is respectively SEQ ID NO.5, SEQ ID NO.6, SEQ
ID NO.7,SEQ ID NO.8.The various combination input of chain I1, I2, I3 are inputted by setting multiple signals, it is glimmering according to normalizing
Light value and threshold decision construct majority logic gate；By setting signal input chain I1 whether input, signal input chain I2
It both may make up AND logic gate as parallel input with signal input chain I3 or may make up OR logic gate；It is pre- in signal input chain I2
Under first input condition, chain I1 is inputted by setting signal and its complementary signal inputs the various combination input of chain I1 ', according to normalizing
Change fluorescent value and threshold decision sets up INHIBIT logic gate；It is defeated by the various combination that multiple signals are inputted chain I1, I1 ', I2
Enter, IHIBIT-AND combinational logic gate is constructed according to normalization fluorescent value and threshold decision output signal.
Operating in 100 μ L reaction systems for the Molecular Logic Gates carries out, and DNA nanotripods use final concentration of
40nM, graphene oxide use final concentration of 20.0ug/mL, and signal inputs chain and uses final concentration of 200nM.
Two, a kind of interaction for regulating and controlling fluorescent small molecule and graphene oxide based on DNA nanotripods constructs molecule
The method of logic gate:
Fluorophor ROX, which is connected, first with the end modified 5 ' ends in center chain C DNA forms modification center chain C-5 '-
ROX recycles DNA nanotechnology to assemble a DNA nanotripods (DNA nanotripod)；With the single-stranded of nanotripods
DNA leg identifies multiple single-stranded DNA sequences as signal input chain, multiple single-stranded DNA sequences are as letter as signal reception chain
Number input chain respectively with three signal reception chain Complementary hybridizations；
When the signal reception chain of DNA nanotripods does not receive signal input chain, i.e., do not hybridize with signal input chain
When be when being single-stranded it is single-stranded, surface of graphene oxide can be adsorbed in；When the signal reception chain and signal of DNA nanotripods input
After chain hybridization, that is, receives signal input chain and form double-strand, from surface of graphene oxide desorption；Chain is inputted by signal
Whether addition controls signal reception chain corresponding on DNA nanotripods in the absorption or desorption of surface of graphene oxide
It is attached；
When unlike signal input chain is inputted with various combination, make DNA nanotripods on the surface of graphene oxide
State is different, so that the interaction of the fluorophor ROX and graphene oxide that are loaded on regulating DNA nanotripods are not
Together, different fluorescence signals is generated, the building of different kinds of molecules logic gate is realized.
The DNA nanotripods assemble preparation in the following ways: taking 1 × TAE-Mg2+Buffer solution is as reaction
Buffer solution, buffer solution pH are 8.0, the EDTA's and 12.5mM of the acetic acid, 2mM of the Tris comprising 40mM, 20mM
Mg(CH3COO)2；Modification center chain C-5 '-ROX and three signal reception chain L1, L2, L3 are added in reaction buffer solution,
It mixes in PCR pipe by the molar ratio of 1:1:1:1, is handled using PCR instrument anneal according to following annealing process: 95 DEG C/
5min, 65 DEG C/30min, 50 DEG C/30min, 37 DEG C/30min, 22 DEG C/30min, 4 DEG C/30min.
Obtain DNA nanotripods after annealing and store liquid, concentration 200nM, 4 DEG C be kept in dark place it is spare.
In the DNA nanotripods, three signal reception chain L1, L2, L3 are converted into center chain C-5 '-ROX is modified
One equilateral triangle, the 3 ' ends and 5 ' ends of center chain C-5 '-ROX are hybridizing the double stranded section middle to be formed congregation with L2,
And a notch is formed in phosphoric acid backbone；
In the DNA nanotripods, a side section of three signal reception chains L1, L2, L3 respectively with modification center
Three side complementary pairings of the equilateral triangle of chain C-5 '-ROX hybridize, and three signal reception chains L1, L2, L3 are connected to center
After chain, three DNA double chains Bian Weisan sections isometric of center chain, length is about 2 spiral recurring units (21 base-pairs
(bp), the T base not hybridized with three signal reception chains L1, L2, L3 is respectively provided with as hinge on three angles of center chain, make
The three segment DNA double-strands on three sides of heart chain form 60 ° of dog-ear two-by-two；The other side part of three signal reception chains L1, L2, L3
Hybridize respectively with multiple signals input chain I1, I2, I3 complementary pairing.
It is as follows that the DNA nanotripods set up specific structure:
Specific structure is as follows after the DNA nanotripods and input chain I1, I2, I3 hybridization of multiple signals:
DNA sequence dna designed by the present invention is as follows:
Multiple single-stranded DNA sequences of the invention are assembled into the special knot of DNA nanotripods (DNA nanotripod)
Structure, the interaction of multiple single stranded DNA legs mutual cooperation regulation fluorophor ROX and graphene oxide, while realizing a variety of points
The building of sub- logic gate.
The Molecular Logic Gates of building include three input majority logic gates, two input AND logic gates, two input OR logics
Door, two input INHIBIT logic gates and three input IHIBIT-AND combinational logic gates；
1, the various combination that chain I1, I2, I3 are inputted by setting multiple signals inputs, according to normalization fluorescent value and threshold value
Judge that output signal sets up majority logic gate；
2, input whether chain I1 inputs by presetting signal, signal inputs chain I2 and signal input chain I3 as flat
Row input both may make up AND logic gate or may make up OR logic gate: at signal input chain I1 not input condition, be believed by setting
The various combination input of number input chain I2, I3, set up AND logic gate；When signal input chain I1 is pre-entered, by setting
The various combination input for determining signal input chain I2, I3, sets up OR logic gate；
If being added without signal input chain I1, i.e. signal input chain I1 input is set as " 0 ", then signal input chain I2 and letter
Number input chain I3 constitutes AND logic gate；If pre-entering signal chains I1, i.e. signal input chain I1 input is set as " 1 ", then believes
Number input chain I2 and signal input chain I3 constitute OR logic gate；Thus it is able to achieve and inputs the defeated of chain I1 by presetting signal
Enter, signal input chain I2 is made both to have may make up AND logic gate as parallel input with signal input chain I3 or may make up OR logic
Door.
3, when signal input chain I2 is pre-entered, chain I1 and its complementary strand I1 ' are inputted not by setting signal
With combination input, INHIBIT logic gate can be set up according to normalization fluorescent value and threshold decision:
Complementary signal, which inputs chain I1 ', to input chain I1 ' and letter in complementary signal with signal input chain I1 complementary pairing hybridization
In the case that number input chain I1 is existed simultaneously, signal, which inputs chain I1, more preferentially to input chain with signal compared to signal reception chain
I1 ' hybridization, forms the double-stranded DNA of complete complementary pairing, and signal input chain I1 can not be hybridized with signal reception chain, i.e.,
Signal input chain I1 can not input the signal path of nanotripods.
4, the various combination that chain I1, I1 ', I2 are inputted by setting multiple signals inputs, according to normalization fluorescent value and threshold
Value judges that output signal constructs IHIBIT-AND combinational logic gate.
Specific implementation are as follows:
1, the operation of three input majority logic gates.Be added in EP pipe 20 μ L DNA nanotripod storage liquid,
50μL H2O and 10 μ L graphene oxide water solutions mix, and are incubated for 10min under room temperature.By each signal input chain I1, I2 and
I3, be added separately to or combination of two be added or three be all added, then with water by response sample volume polishing extremely
100 μ L, make the final concentration of 40nM of DNA nanotripod in system, and each signal inputs the final concentration of 200nM of chain.Sample is mixed
It is even, and in 37 DEG C of incubation 30min.Later, it takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.Fluorescence measurement
Use QuantaMasterTMSepectrophotofluorometer (Canada), excitation wavelength and launch wavelength be respectively set to 588 nm and
603nm, exciting slit width and transmite slit width are respectively set to 5nm and 5nm.20 μ L DNA nanotripod are taken to store
Liquid is diluted with water to 100 μ L and measures its fluorescence, mean value is taken, and mean value is classified as 1.0, then as standard, by each sample
Fluorescent value do normalized." 1 " and " 0 ", normalized fluorescent value 0.60 are respectively defined as whether inputting the addition of DNA chain
It is set to threshold value, it is " 0 " that logic output is defined when fluorescent value is less than 0.60, and fluorescent value defines logic output when being greater than 0.60 be " 1 ".
2, the operation of other each logic gates and combinational logic gate inputs chain using corresponding signal, at operating method and data
Reason mode is identical as majority logic gate.
3, preferably, DNA nanotripods use final concentration of 40nM.
4, preferably, each signal input chain uses final concentration of 200nM.
5, preferably, graphene oxide uses final concentration of 20.0ug/mL.
The solution have the advantages that:
DNA nanotripods are assembled using DNA nanotechnology and as bracket, it is negative that it is regulated and controled on nanoscale
The fluorophor ROX of load and the interaction of graphene oxide construct the majority logic gate of one three input.
Three input majority logic gates of building can be converted between OR logic gate and AND logic gate.
The molecular computing module being made of DNA nanotripods and graphene oxide has multifunctionality, this multi-functional
Property become one building Molecular Logic Gates universal module, can further construct INHIBIT logic gate.
INHIBIT logic gate and AND logic gate can combine the INHIBIT-AND combinational logic gate for constituting three inputs.
Detailed description of the invention
Fig. 1 is the fluorophor ROX and oxidation stone that DNA nanotripods of the present invention regulate and control its load on nanoscale
The interaction principle figure of black alkene.
Fig. 2 is that the assembling of polyacrylamide gel electrophoresis validating DNA nanotripods and three of them single-stranded signal receive and lead to
Identification of the road to signal input chain.
Fig. 3 is to input majority logic gate using I1, I2 and I3 as the three of input signal.
Fig. 4 is to input AND logic gate and OR logic gate using I2 and I3 as the two of input signal.
Fig. 5 is to input INHIBIT logic gate using I1 and I1' as the two of input signal.
Fig. 6 is to input INHIBIT-AND combinational logic gate using I1, I1' and I2 as the three of input signal.
Specific embodiment
Below with reference to embodiment, the invention will be further described, but the present invention is not limited to following embodiments.
The embodiment of the present invention is as follows:
The DNA nanotripods in each embodiment assemble preparation in the following ways below: containing 1 × TAE-Mg2+
Buffer solution (40mM Tris, pH 8.0,20mM acetic acid, 2mM EDTA, 12.5mM Mg (CH3COO)2) reaction
In system, modification center chain C-5 '-ROX is mixed in PCR pipe with L1, L2 and L3 by the molar ratio of 1:1:1:1, is then made
With PCR instrument anneal, assemble nanometer tripod, specific annealing process is 95 DEG C/5min, 65 DEG C/30min, 50 DEG C/30min,
37 DEG C/30min, 22 DEG C/30min, 4 DEG C/30min.Three foot of DNA nanometer is confirmed using 12% polyacrylamide gel electrophoresis
The assembling of frame, and ability of its three single stranded DNA legs as signal receiving channel identification signal input chain is verified, as a result such as Fig. 2
It is shown.Assembled DNA nanotripods storage liquid concentration is 200 nM, 4 DEG C be kept in dark place it is spare.
The base sequence of the center chain C-5 '-ROX of the DNA nanotripods of embodiment is SEQ ID NO.1, three signals
The base sequence of reception chain L1, L2, L3 are respectively SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.
Embodiment 1
Regulate and control the interaction of the fluorophor ROX and graphene oxide of its load, building using DNA nanotripods
Majority logic gate.
8 EP pipes are taken, number is 1,2,3,4,5,6,7 and 8 respectively, and the DNA that 20 μ L concentration are 200nM is added thereto
Nanotripod stores liquid, 50 μ L H2The graphene oxide water that O and the good concentration of the 10 preparatory ultrasonic disperses of μ L are 200.0ug/mL
Solution mixes, and is incubated for 10min under room temperature.
Then, it plus signal input chain or chain I1, I2 and I3 is not input a signal into is added separately to or combination of two
It is added or is all added, the single-stranded leg of tripod is used as signal receiving channel, receives corresponding signal and inputs chain and therewith
Hybridization form double-strand, get off from surface of graphene oxide desorption, make DNA receive tripod surface of graphene oxide state not
Together, to regulate and control the interaction of the fluorophor and graphene oxide of its load, different fluorescence signals is provided.Specific processing
It is as follows.
20 μ L H are added into No. 1 pipe2O is mixed；I1 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 2 pipes2O,
It mixes；I2 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 3 pipes2O is mixed；It is 5 μM that 4 μ L concentration are added into No. 4 pipes
I3 chain and 16 μ L H2O is mixed；The I2 chain and 12 μ that I1 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are added into No. 5 pipes
L H2O is mixed；The I3 chain and 12 μ L H that I1 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are added into No. 6 pipes2O is mixed；
The I3 chain and 12 μ L H that I2 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are added into No. 7 pipes2O is mixed；Into No. 8 pipes
The I3 chain and 8 μ L H that I2 chain, 4 μ L concentration that I1 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are 5 μM is added2O is mixed.
Various kinds quality control is placed in biochemical cultivation case, 37 DEG C of incubation 30min.
It takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.
Each sample is done 3 times in parallel.
The measurement result of each sample is normalized and is mapped, as a result as shown in Fig. 3 (a), No. 1, No. 2, No. 3
It is below 0.6 with the normalization fluorescent value of No. 4 pipes, is exported as " 0 "；The normalization fluorescent value of No. 5, No. 6, No. 7 and No. 8 pipes is high
In 0.6, export as " 1 ".Shown in the truth table of logical operation such as Fig. 3 (b), which meets three input majority logics
Door.The result shows that the method according to the invention, successfully constructs majority logic gate, Fig. 3 (c) is three input
The circuit diagram of majority logic gate.
In this embodiment, the base sequence of multiple signal input chain I1, I2, I3 are respectively SEQ ID NO.5, SEQ ID
NO.6, SEQ ID NO.7, the i.e. sequence of I1 are GGTTCTGAATGCTGAC；The sequence of I2 is CCAATCCTTCGAGATG；I3
Sequence be ATCGCTAGTGAGGGTA.
Embodiment 2
The building of two input AND logic gates and OR logic gate
4 EP pipes are taken, number is 1,2,3 and 4 respectively, and the DNA that 20 μ L concentration are 200nM is added thereto
Nanotripod stores liquid, 50 μ L H2The graphene oxide that O and the good concentration of the 10 preparatory ultrasonic disperses of μ L are 200.0 ug/mL
Aqueous solution mixes, and is incubated for 10min under room temperature, then does following processing.
20 μ L H are added into No. 1 pipe2O is mixed；I2 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 2 pipes2O,
It mixes；I3 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 3 pipes2O is mixed；It is 5 μM that 4 μ L concentration are added into No. 4 pipes
I2 chain, 4 μ L concentration be 5 μM of I3 chain and 12 μ L H2O is mixed.
Various kinds quality control is placed in biochemical cultivation case, 37 DEG C of incubation 30min.
It takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.
Each sample is done 3 times in parallel.
The measurement result of each sample is normalized and is mapped, as a result as shown in Fig. 4 (a), No. 1, No. 2 and No. 3
The normalization fluorescent value of pipe is below 0.6, exports as " 0 "；The normalization fluorescent value of No. 4 pipes is higher than 0.6, exports as " 1 ".As a result
Show the method according to the invention, successfully constructs two input AND logic gates.
4 EP pipes are separately taken, number is 1,2,3 and 4 respectively, and the DNA that 20 μ L concentration are 200nM is added thereto
Nanotripod stores I1 chain, the 46 μ L H of liquid, 4 μ L concentration for 5 μM2O and the good concentration of the 10 preparatory ultrasonic disperses of μ L are
The graphene oxide water solution of 200.0ug/mL mixes, and is incubated for 10min under room temperature, then does following processing.
20 μ L H are added into No. 1 pipe2O is mixed；I2 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 2 pipes2O,
It mixes；I3 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 3 pipes2O is mixed；It is 5 μM that 4 μ L concentration are added into No. 4 pipes
I2 chain, 4 μ L concentration be 5 μM of I3 chain and 12 μ L H2O is mixed.
Various kinds quality control is placed in biochemical cultivation case, 37 DEG C of incubation 30min.
It takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.
Each sample is done 3 times in parallel.
The measurement result of each sample is normalized and is mapped, as a result as shown in Fig. 4 (b), the normalization of No. 1 pipe
Fluorescent value is lower than 0.6, exports as " 0 "；, No. 2, the normalization fluorescent values of No. 3 and No. 4 pipes be above 0.6, export as " 1 ".As a result
Show the method according to the invention, successfully constructs two input OR logic gates.
In this embodiment, if I1 input is set as " 0 ", I2 and I3 constitute AND logic gate；If I1 input set
It is set to " 1 ", I2 and I3 constitute OR logic gate.That is: by presetting the input of I1, I2 and I3 may make up AND logic gate or
OR logic gate.
The sequence of I1, I2 and I3 are the same as embodiment 1 in this embodiment.
Embodiment 3
The building of INHIBIT logic gate
4 EP pipes are taken, number is 1,2,3 and 4 respectively, and the DNA that 20 μ L concentration are 200nM is added thereto
Nanotripod stores I2 chain, the 46 μ L H of liquid, 4 μ L concentration for 5 μM2O and the good concentration of the 10 preparatory ultrasonic disperses of μ L are
The graphene oxide water solution of 200.0ug/mL mixes, and is incubated for 10min under room temperature, then does following processing.
20 μ L H are added into No. 1 pipe2O is mixed；I1 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 2 pipes2O,
It mixes；I1' chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 3 pipes2O is mixed；It is 5 μ that 4 μ L concentration are added into No. 4 pipes
The I1 chain of M, the I1' chain and 12 μ L H that 4 μ L concentration are 5 μM2O is mixed.
Various kinds quality control is placed in biochemical cultivation case, 37 DEG C of incubation 30min.
It takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.
Each sample is done 3 times in parallel.
The measurement result of each sample is normalized and is mapped, as a result as shown in figure 5, No. 1 pipe, No. 3 pipes and 4
The normalization fluorescent value of number pipe is lower than 0.6, exports as " 0 "；The normalization fluorescent value of No. 2 pipes is above 0.6, exports as " 1 ".Knot
Fruit shows the method according to the invention, successfully constructs two input INHIBIT logic gates.
In this embodiment, the sequence of I1, I2 are the same as embodiment 1；The base sequence of multiple signal input chain I1 is SEQ ID
NO.5, the i.e. sequence of I1 are GGTTCTGAATGCTGAC；The base sequence that complementary signal inputs chain I1 is SEQ ID NO.8, i.e.,
The sequence of I1' is GTCAGCATTCAGAACC.
Embodiment 4
The building of three input INHIBIT-AND combinational logic gates
8 EP pipes are taken, number is 1,2,3,4,5,6,7 and 8 respectively, and the DNA that 20 μ L concentration are 200nM is added thereto
Nanotripod stores liquid, 50 μ L H2The graphene oxide water that O and the good concentration of the 10 preparatory ultrasonic disperses of μ L are 200.0ug/mL
Solution mixes, and is incubated for 10min under room temperature, then does following processing.
20 μ L H are added into No. 1 pipe2O is mixed；I1 chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 2 pipes2O,
It mixes；I1' chain and 16 μ L H that 4 μ L concentration are 5 μM are added into No. 3 pipes2O is mixed；It is 5 μ that 4 μ L concentration are added into No. 4 pipes
The I1 chain of M, the I1' chain and 12 μ L H that 4 μ L concentration are 5 μM2O is mixed；Be added into No. 5 pipes 4 μ L concentration be 5 μM I2 chain and
16μL H2O is mixed；The I2 chain and 12 μ L H that I1 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are added into No. 6 pipes2O is mixed
It is even；The I2 chain and 12 μ L H that I1' chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are added into No. 7 pipes2O is mixed；To No. 8
The I2 chain and 8 μ L H that I1' chain, 4 μ L concentration that I1 chain, 4 μ L concentration that 4 μ L concentration are 5 μM are 5 μM are 5 μM are added in pipe2O,
It mixes.
Various kinds quality control is placed in biochemical cultivation case, 37 DEG C of incubation 30min.
It takes 95 μ L samples to be transferred in cuvette and measures fluorescent under room temperature.
Each sample is done 3 times in parallel.
The measurement result of each sample is normalized and is mapped, as a result as shown in Fig. 6 (a), No. 1, No. 2, No. 3,4
Number, the normalization fluorescent values of No. 5, No. 7 and No. 8 pipes be below 0.6, export as " 0 "；The normalization fluorescent value of No. 6 pipes is higher than
0.6, it exports as " 1 ", shown in the truth table of logical operation such as Fig. 6 (b).The result shows that the method according to the invention successfully constructs
Three input INHIBIT-AND combinational logic gates.
The sequence of I1, I1' and I2 are the same as embodiment 3 in this embodiment.
The gene order being related in the present invention is as follows:
SEQ ID NO.1: the base sequence of center chain C
Source: artificial synthesized
AGGCACCATCGTGGAGTGTCACGGTGCATGGCTTGCGACTGCTACGG ACGCTCTCTAGGTCTTGCC
SEQ ID NO.2: the base sequence of signal reception chain L1
Source: artificial synthesized
AGCCATGCACCGTGACACTCCTGTCAGCATTCAGAACC
SEQ ID NO.3: the base sequence of signal reception chain L2
Source: artificial synthesized
CGATGGTGCCTGGCAAGACCTTCATCTCGAAGGATTGG
SEQ ID NO.4: the base sequence of signal reception chain L3
Source: artificial synthesized
GAGAGCGTCCGTAGCAGTCGCTTACCCTCACTAGCGAT
SEQ ID NO.5: the base sequence of signal input chain I1
Source: artificial synthesized
GGTTCTGAATGCTGAC
SEQ ID NO.6: the base sequence of signal input chain I2
Source: artificial synthesized
CCAATCCTTCGAGATG
SEQ ID NO.7: the base sequence of signal input chain I3
Source: artificial synthesized
ATCGCTAGTGAGGGTA
SEQ ID NO.8: the base sequence of complementary signal input chain I1 '
Source: artificial synthesized
GTACGACTCACCAATCCTTCGAGATGAACCGACGTGACAT
Sequence table
<110>Zhejiang Academy of Agricultural Science
<120>based on DNA nanotripods regulation fluorescent small molecule and graphene oxide interaction Molecular Logic Gates and its
Construction method
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aggcaccatc gtggagtgtc acggtgcatg gcttgcgact gctacggacg ctctctaggt 60
cttgcc 66
<210> 2
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
agccatgcac cgtgacactc ctgtcagcat tcagaacc 38
<210> 3
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cgatggtgcc tggcaagacc ttcatctcga aggattgg 38
<210> 4
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagagcgtcc gtagcagtcg cttaccctca ctagcgat 38
<210> 5
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ggttctgaat gctgac 16
<210> 6
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccaatccttc gagatg 16
<210> 7
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atcgctagtg agggta 16
<210> 8
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gtacgactca ccaatccttc gagatgaacc gacgtgacat 40
Claims (10)
1. a kind of Molecular Logic Gates based on DNA nanotripods regulation fluorescent small molecule and graphene oxide interaction,
It is characterized in that: being received including graphene oxide, DNA nanotripods (DNA nanotripod) and multiple single-stranded DNA sequences, DNA
Rice tripod includes a center chain and three side chains, and a center chain passes through in a part hybridization formation therein of three side chains
Heart triangle, the remainder of three side chains extend out to form single stranded DNA leg from center Atria vertex respectively, utilize
DNA is end modified to make center chain load fluorophor ROX, multiple using the single stranded DNA leg of nanotripods as signal reception chain
Single-stranded DNA sequence as signal input chain respectively with three signal reception chain Complementary hybridizations；Signal reception chain is defeated not with signal
Be adsorbed in surface of graphene oxide when entering chain hybridization, signal reception chain hybridize with signal input chain become double-strand after from aoxidizing stone
Black alkene surface desorption is attached.
2. according to claim 1 a kind of mutual based on DNA nanotripods regulation fluorescent small molecule and graphene oxide
The Molecular Logic Gates of effect, it is characterised in that: the base sequence of the center chain of the DNA nanotripods is SEQ ID
NO.1, the base sequence of three signal reception chains are respectively SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.
3. according to claim 2 a kind of mutual based on DNA nanotripods regulation fluorescent small molecule and graphene oxide
The Molecular Logic Gates of effect, it is characterised in that: the DNA nanotripods package assembly is as follows:
4. according to claim 1 a kind of mutual based on DNA nanotripods regulation fluorescent small molecule and graphene oxide
The Molecular Logic Gates of effect, it is characterised in that: multiple signal input chains are that three signals input chain I1, I2, I3, base sequence point
It Wei not SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7；The difference of chain I1, I2, I3 are inputted by setting multiple signals
Combination input, constructs majority logic gate；Either by preset signal input chain I1 whether input, signal input
Chain I2 both may make up AND logic gate as parallel input with signal input chain I3 or may make up OR logic gate.
5. according to claim 1 a kind of mutual based on DNA nanotripods regulation fluorescent small molecule and graphene oxide
The Molecular Logic Gates of effect, it is characterised in that:
Multiple signal input chains are that signal inputs chain I2, signal inputs chain I1 and complementary signal inputs chain I1 ', complementary signal input
The base sequence of chain I1 ' is SEQ ID NO.8；When signal input chain I2 is pre-entered, chain is inputted by setting signal
The various combination input of I1 and complementary signal input chain I1 ', set up INHIBIT logic according to normalization fluorescent value and threshold decision
Door.
6. according to claim 1 a kind of mutual based on DNA nanotripods regulation fluorescent small molecule and graphene oxide
The Molecular Logic Gates of effect, it is characterised in that: operating in 100 μ L reaction systems for the Molecular Logic Gates carries out, and DNA receives
Rice tripod uses final concentration of 40nM, and graphene oxide uses final concentration of 20.0ug/mL, and signal inputs chain and uses final concentration
For 200nM.
7. a kind of interaction for regulating and controlling fluorescent small molecule and graphene oxide based on DNA nanotripods constructs Molecular Logic Gates
Method, it is characterised in that: first with the end modified 5 ' ends in center chain C DNA connect fluorophor ROX formed modification in
Heart chain C-5 '-ROX, then assemble a DNA nanotripods (DNA nanotripod)；With the single stranded DNA leg of nanotripods
As signal reception chain, multiple single-stranded DNA sequences as signal input chain are identified；
When the signal reception chain of DNA nanotripods does not receive signal input chain, i.e., it is when not hybridizing with signal input chain
It is single-stranded, surface of graphene oxide can be adsorbed in；After the signal reception chain of DNA nanotripods hybridizes with signal input chain, i.e.,
It receives signal input chain and forms double-strand, from surface of graphene oxide desorption；By signal input chain whether addition control
Absorption or desorption of the corresponding signal reception chain in surface of graphene oxide on DNA nanotripods processed；
When unlike signal input chain is inputted with various combination, make DNA nanotripods in the state on the surface of graphene oxide
Difference produces so that the fluorophor ROX loaded on regulating DNA nanotripods is different from the interaction of graphene oxide
Raw different fluorescence signal realizes the building of different kinds of molecules logic gate.
8. a kind of phase based on DNA nanotripods regulation fluorescent small molecule and graphene oxide according to claim 7
The method of interaction building Molecular Logic Gates, it is characterised in that:
The DNA nanotripods assemble preparation in the following ways: taking 1 × TAE-Mg2+Buffer solution is as reaction buffering
Solution, buffer solution pH are 8.0, the acetic acid of the Tris comprising 40mM, 20mM, the Mg (CH of the EDTA and 12.5mM of 2mM3COO)2；
Modification center chain C-5 '-ROX and three signal reception chain L1, L2, L3 are added in reaction buffer solution, by 1:1:1:1's
Molar ratio mixes in PCR pipe, is handled using PCR instrument anneal according to following annealing process: 95 DEG C/5min, 65 DEG C/
30min, 50 DEG C/30min, 37 DEG C/30min, 22 DEG C/30min, 4 DEG C/30min.
9. a kind of phase based on DNA nanotripods regulation fluorescent small molecule and graphene oxide according to claim 7
The method of interaction building Molecular Logic Gates, it is characterised in that:
In the DNA nanotripods, three signal reception chain L1, L2, L3 are converted into one for center chain C-5 '-ROX is modified
Equilateral triangle makes the end center chain C3 ' for modifying center chain C-5 '-ROX and 5 ' ends merge in the middle of signal reception chain L2,
And a notch is formed in phosphoric acid backbone；
In the DNA nanotripods, a side section of three signal reception chains L1, L2, L3 respectively with modification center chain C-
Three side complementary pairings of the equilateral triangle of 5 '-ROX hybridize, after three signal reception chains L1, L2, L3 are connected to center chain,
Three DNA double chains Bian Weisan sections isometric of center chain, length are about 2 spiral recurring units (21 base-pairs (bp), center
It is respectively provided with the T base not hybridized with three signal reception chains L1, L2, L3 on three angles of chain as hinge, makes center chain three
The three segment DNA double-strands on side form 60 ° of dog-ear two-by-two；The other side part of three signal reception chains L1, L2, L3 identify more respectively
A signal inputs chain.
10. a kind of phase based on DNA nanotripods regulation fluorescent small molecule and graphene oxide according to claim 7
The method of interaction building Molecular Logic Gates, it is characterised in that:
The Molecular Logic Gates of building include majority logic gate, two input AND logic gates, two input OR logic gates, two inputs
INHIBIT logic gate and three input IHIBIT-AND combinational logic gates；
1, the various combination that chain I1, I2, I3 are inputted by setting multiple signals inputs, according to normalization fluorescent value and threshold decision
Output signal sets up majority logic gate；
2, input whether chain I1 inputs by presetting signal, signal inputs chain I2 with signal input chain I3 as parallel input
Both it may make up AND logic gate or may make up OR logic gate: at signal input chain I1 not input condition, having been inputted by setting signal
The various combination of chain I2, I3 input, and set up AND logic gate；When signal input chain I1 is pre-entered, pass through setting signal
The various combination input for inputting chain I2, I3, sets up OR logic gate；
3, in the case where signal inputs chain I2 input condition, the various combination for inputting chain I1 and its complementary strand I1 ' by setting signal is defeated
Enter, can set up INHIBIT logic gate according to normalization fluorescent value and threshold decision: complementary signal inputs chain I1 ' can be defeated with signal
Enter the hybridization of chain I1 complementary pairing, in the case where complementary signal input chain I1 ' and signal input chain I1 are existed simultaneously, signal input
Chain I1 can more preferentially hybridize with signal input chain I1 ' compared to signal reception chain, form the double-stranded DNA of complete complementary pairing,
And signal input chain I1 can not be hybridized with signal reception chain.
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