CN103865922A

CN103865922A - Preparation method for RNA

Info

Publication number: CN103865922A
Application number: CN201410113861.0A
Authority: CN
Inventors: 梁兴国; 王星宇
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2014-03-25
Filing date: 2014-03-25
Publication date: 2014-06-18

Abstract

The invention provides a preparation method for RNA. The preparation method comprises the following steps of connecting a linear DNA template into a ring to form annular DNA under the action of short-chain DNA and DNA ligase; synthesizing long-chain RNA in a rolling circle amplification way by utilizing RNA polymerase, wherein the long-chain RNA contains a tandem repeat sequence of required RNA sequences; cutting tandem products on specific sites to generate a required sequence by utilizing a specific nucleic acid sequence and other enzyme or compounds. According to the preparation method, the RNA synthesis process is simple and efficient; moreover, the synthesized RNA sequence has high fidelity, raw material waste is greatly reduced in the process, complex modification cutting over the product is not required, the method is economical and particularly suitable for the large-scale synthesis of small RNA.

Description

The preparation method of a kind of RNA

Technical field

The invention belongs to field of molecular biotechnology, relate in particular to the preparation method of a kind of RNA.

Background technology

Existing RNA synthetic technology all adopts solid state chemistry synthetic, compared with DNA synthetic, need to carry out special protection to 2 '-OH, after end of synthesis, unifiedly again 2 '-OH is carried out to deprotection, has caused so higher RNA to synthesize cost.Meanwhile, RNA sequence in building-up process, Yeast Nucleic Acid of every interpolation, optimum product yield also only between 97～99%, for long RNA sequence, for example, 100ntRNA, yield, below 10%, is generally a kind of waste to ribonucleoside acid starting material.

Except solid state chemistry is synthetic, existing RNA synthetic technology also comprises that the enzyme process RNA that promotor causes synthesizes, although the method can overcome some intrinsic problems that chemosynthesis exists, but its synthetic promoter sequence that must rely on double-stranded DNA form, and RNA sequence synthetic had to Preference, and the RNA combined coefficient that 5 ' end is rich in purine is far above purine-containing sequence not.Meanwhile, the enzyme process RNA preparation that promotor relies on needs to use the enzymes such as DNAzyme to cut Primary product, and the cutting of DNAzyme also has sequence Preference, and the RNA sequence that can cut is limited, therefore can be also limited by the synthetic RNA kind of the method.In addition, the efficiency of DNAzyme cutting is not as good as proteolytic enzyme, and is easily subject to the impact of catalysis microenvironment.Above problem has hindered the enzyme process RNA preparation of promotor dependence as the replacement method of chemosynthesis RNA.

Summary of the invention

The object of the present invention is to provide the preparation method of a kind of RNA, be intended to solve the existing problems and shortcomings of prior art.

The present invention is achieved in that the preparation method of a kind of RNA, comprises the following steps:

(1) cDNA of design and object RNA complementation connects into ring by described cDNA under the effect of short chain DNA and DNA ligase, forms cyclic DNA;

(2) utilize RNA polymerase, synthesize long-chain RNA with the form of rolling circle amplification, the tandem repetitive sequence that this long-chain RNA contains required RNA sequence;

(3) utilize specific nucleotide sequence and other enzyme or compound, the product of connecting, in the cutting of specificity site, generates required sequence.

Preferably, in step (1), the head and the tail sequence of described short chain DNA sequence dna and cDNA chain is carried out base complementrity pairing, and makes cDNA end to end, forms ring-type cDNA.

Preferably, described short chain DNA length is 11～15nt.

Preferably, in step (2), described RNA polymerase is t7 rna polymerase, E.coliRNA polysaccharase or Syn5RNA polysaccharase.

Preferably, in step (3), described specific nucleotide sequence be oxygen methylate modify short chain DNA sequence dna, described DNA sequence dna with RNA product in the combination of desired location complementary pairing, and under the effect of RNaseH, at specific site cutting RNA, obtain object RNA sequence.

The present invention overcomes the deficiencies in the prior art, and the preparation method of a kind of RNA is provided, and by linear cDNA template is connected into ring under the effect of short chain DNA and DNA ligase, forms ring-type cDNA; Utilize RNA polymerase, synthesize long-chain RNA with the form of rolling circle amplification, the tandem repetitive sequence that contains required RNA sequence; Finally utilize specific nucleotide sequence and other enzyme or compound, the product of connecting, in the cutting of specificity site, generates required sequence.

In the present invention by rolling circle amplification (RCA) is combined to prepare RNA with RNaseH enzyme incision technology, wherein, RCA is take cyclic DNA as template, by a short DNA/RNA primer (with the complementation of part circular template), under enzyme catalysis, NTPs is transformed into singlestranded RNA, product strand comprises hundreds and thousands of the complementary fragments of the template repeating.This method synthesizing single-stranded RNA that can directly increase.RCA was used for amplification of signal in the past and detected target nucleic acid molecules, but due to its characteristic that produces strand, can be used for synthesizing preparation RNA.The RNA that the product of RCA amplification is strand is the complementary sequence of template cDNA.Under normal circumstances, RCA can be by template amplification 10 ³～10 ⁴doubly, product is tandem repetitive sequence.On this basis, produce required RNA in conjunction with the method for the rolling circle amplification under RNA polymerase effect and specific RNA shearing, amplification efficiency can reach 10 ⁴～10 ⁵.

It is synthetic that the present invention adopts cyclic DNA template to increase, and do not need the promoter sequence of preparing again double-stranded DNA form to cause amplification.Meanwhile, the tandem repetitive sequence of the object RNA that the product of rolling circle amplification is long-chain, has overcome RNA polymerase 5 ' selectivity and 3 ' end and has added at random the shortcoming of 1～2nt base.Meanwhile, can select several different methods cutting to product.Wherein utilize the nucleotide sequence of modifying to combine the specificity cutting of carrying out RNA with RNaseH, the cutting of RNA is not had to sequence restriction, can synthesize as requested the RNA of any sequence.The catalytic efficiency of RNaseH is higher simultaneously, and is not subject to the impact of pH, temperature and system ionic intensity.

Therefore, RNA building-up process of the present invention is more succinct, efficient, and synthetic RNA sequence has high fidelity, does not produce wastage of material in process, does not need to carry out miscellaneous modification for product again, more economically, is particularly useful for the synthetic of a large amount of little RNA.

Accompanying drawing explanation

Fig. 1 is the preparation method's of RNA of the present invention Overall Steps schematic diagram;

Fig. 2 is that embodiment of the present invention neutral line cDNA becomes ring principle schematic;

Fig. 3 be the short-and-medium chain DNA of the embodiment of the present invention structure with become ring principle schematic;

Fig. 4 is RCA transcription amplification principle schematic in the embodiment of the present invention;

Fig. 5 A is the special cutting long-chain of RNaseH RNA principle schematic in the embodiment of the present invention in the embodiment of the present invention, and 5B is the detail view of the special cutting sequence of RNaseH.

Embodiment

In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.

Embodiment

A preparation method of RNA, as shown in Figure 1, comprises the following steps:

Step (1): the cDNA of design and object RNA complementation, described cDNA is connected into ring under the effect of short chain DNA and DNA ligase, form ring-type cDNA;

(2): ring-type cDNA template, under the effect of t7 rna polymerase, take short chain DNA as primer, causes and rolls ring transcription amplification, form the long-chain RNA of the series connection repetition being formed by target RNA;

(3): the short chain DNA associating that utilizes RNaseH and oxygen methyl to modify, in specific site target, long-chain RNA is cut;

(4): through the RNA long-chain of cutting, obtain object RNA sequence, its molecule number is 10 of template cDNA ⁴～10 ⁵doubly.

In step (1), as shown in Figure 2, more specifically comprise the following steps:

(a) design and synthesize and the cDNA template of target RNA complementation, and become the needed short chain DNA sequence dna of ring, 5 of cDNA ' end carries out phosphorylation modification, so that ligase enzyme connects;

(b) cDNA is mixed with becoming the needed short chain DNA of ring, under the effect of T4DNA ligase enzyme, chain cDNA template is joined end to end, form ring-type cDNA template.

Wherein, short chain DNA sequence dna length is 11～15nt, and the head and the tail sequence of short chain DNA sequence dna and cDNA chain carries out base complementrity pairing, and makes cDNA end to end, forms ring-type cDNA.In actual application, short chain DNA sequence dna length is not particularly limited, as long as can make the end to end one-tenth of cDNA and be stable into the sequence of ring, can serve as short chain DNA.The structure of short chain DNA is illustrated in fig. 3 shown below with becoming ring principle, wherein, black font partial sequence is the one of short chain DNA, it can be with the head and the tail of cDNA grey (shown in glissade) complementation, under the effect of DNA ligase, cDNA is connected into ring-type, its 3 ' end-OH is also as the initial synthetic site of RNA polymerase simultaneously, and initiation is transcribed.

Step (2): utilize RNA polymerase, synthesize long-chain RNA with the form of rolling circle amplification, this long-chain RNA is the tandem repetitive sequence of required RNA sequence composition;

In step (2), as shown in Figure 4, the ring-type cDNA connecting is the template of the synthetic RNA of amplification, and short chain DNA annealing simultaneously becomes local two strands with circular template, the initiation site that its 3 '-OH causes as amplification.In system, add t7 rna polymerase, NTPs, RNA enzyme inhibitors, transcribe as template is initial take ring-type cDNA.T7 rna polymerase, take ring-type cDNA as template, in strict accordance with base complementrity pair principle, participates in NTPs in RNA chain.RNA polymerase is taken turns along shuttering work one, the RNA of a synthetic unit.Due to the strand displacement ability that T7RNA enzyme has, often carry out taking turns amplification, the RNA in the last round of cDNA of being present in template will be replaced, move in circles, final synthetic long-chain RNA, repeats series connection by the RNA unit of multiple same template cDNA complementations and is formed, and multiplicity is 10 ⁴～10 ⁵inferior left and right.

Step (3) is utilized specific nucleotide sequence and other enzyme or compound, and the product of connecting, in the cutting of specificity site, generates required sequence.

In step (3), more specifically, after the synthetic long-chain RNA of step (2) finishes, need to cut product, to obtain the RNA of required length.Cutting RNA has multiple choices, comprising:

(a) the RNaseH associating oxygen short chain DNA sequence dna of modifying that methylates;

(b) DNA of lanthanide metal ion combined modification;

(c) amido modified compounds can be used for cutting the RNA containing circular structure;

(d) DNAzyme cutting.

Through the RNA long-chain of cutting, obtain object RNA sequence, its molecule number is 104～105 times of template cDNA.

Below exemplify a kind of specific embodiments: as shown in Figure 5, wherein, black lines represents the long-chain RNA of transcription amplification, repeated to be composed in series by multiple units, in Fig. 5 A, the short chain DNA sequence dna that utilizes oxygen methyl to modify, annealed combination, to the connected position of long-chain RNA unit, and guides the specific cutting of RNaseH RNA for RNaseH provides the site of identification combination.Fig. 5 B is that oxygen methyl is modified the detail view of short chain DNA sequence dna with cleavage site combination.The Nucleotide that underscore marks sequence carries out the modification of oxygen methyl, all the other common DNA sequence dnas, modification short chain is sequence-specific is attached to long-chain RNA above, instructs RNaseH simultaneously, cut in arrow indication position (between UG), the product obtaining is just object RNA.Through after RNaseH cutting, long-chain RNA is cut into the RNA of expection length, and this RNA at 5 ' end with phosphate group, in full accord with natural little RNA structure.And the RNA5 ' of chemosynthesis holds as-OH, the in the situation that of needs phosphorylation, adding phosphate group cost with chemical method can increase much conventionally.Use present method, there is the function of autophosphorylation RNA.Enzyme is cut the separation through high performance liquid chromatography later, and purifying, and lyophilize, just can obtain the on all four RNA of same native state.

Than the shortcoming and defect of prior art, the present invention has following beneficial effect: RNA building-up process of the present invention is more succinct, efficient, and synthetic RNA sequence has high fidelity, in process, greatly reduce wastage of material, do not need to carry out again miscellaneous modification cutting for product, more economically, be particularly useful for the synthetic of a large amount of little RNA.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims

1. a preparation method of RNA, is characterized in that comprising the following steps:

(1) cDNA of design and object RNA complementation connects into ring by described cDNA under the effect of short chain DNA and DNA ligase, forms ring-type cDNA;

2. the preparation method of RNA as claimed in claim 1, is characterized in that, in step (1), the head and the tail sequence of described short chain DNA sequence dna and cDNA chain is carried out base complementrity pairing, and makes cDNA end to end, forms ring-type cDNA.

3. the preparation method of RNA as claimed in claim 2, is characterized in that, described short chain DNA length is 11～15nt.

4. the preparation method of RNA as claimed in claim 3, is characterized in that, in step (2), described RNA polymerase is t7 rna polymerase, E.coliRNA polysaccharase or Syn5RNA polysaccharase.

5. the preparation method of RNA as claimed in claim 4, it is characterized in that, in step (3), described specific nucleotide sequence be oxygen methylate modify short chain DNA sequence dna, described DNA sequence dna with RNA product in the combination of desired location complementary pairing, and under the effect of RNaseH, at specific site cutting RNA, obtain object RNA sequence.