WO2005118807A1

WO2005118807A1 - Process for creating a double-stranded polyribonucleotide sequence with terminal overhang, as well as a process for creating a double-stranded polynucleotide construct and an application

Info

Publication number: WO2005118807A1
Application number: PCT/NL2005/000401
Authority: WO
Inventors: Nynke Hester Dekker; Peter Veenhuizen
Original assignee: Technische Universiteit Delft
Priority date: 2004-06-04
Filing date: 2005-06-01
Publication date: 2005-12-15
Also published as: NL1026335C2

Abstract

The invention relates to a process for creating double-stranded RNA (16) having a terminal overhang. In accordance with the invention a DNA amplification is used for this purpose, followed by a transcription of the amplified DNA. When amplifying the DNA, primer pairs are used and care is taken that additional sequences (18, 19) are present that will ultimately provide the terminal overhangs.

Description

Process for preparing a double-stranded polyribonucleotide sequence with terminal overhang, as well as a process for preparing a double-stranded polynucleotide construct and an application

The present invention relates to a process for preparing a double-stranded polyribonucleotide sequence with a terminal overhang. Recent years have shown an increasing awareness re- garding the importance of the role that double-stranded RNA plays in nature, as well as in research and in the development of medical treatments. Double-stranded RNA molecules are of interest for various applications, such as RNA interference experiments and the preparation of polynucleic acid con- structs. In DNA, overlapping and complementary terminal overhangs can be prepared with the aid of restriction enzymes, which nature supplies in abundance, as well as in a wide variety. Such luxury does not exist with respect to RNA. Accordingly, there is an urgent need for a technique with which it is possible to form double-st.randed RNA with at least 1 terminal overhang. It is the object of the present invention, to provide a process with which such RNA can be formed in a simple manner, and using techniques that are not new in themselves. To this end, the process according to the invention is characterized by starting from two complementary single- stranded DNA sequences, designated first DNA sequence and second DNA sequence

— wherein the first primer of a first primer pair comprising a first primer and a second primer, is contacted with the first DNA sequence, and the second primer is contacted with the second DNA sequence that is complementary to the first DNA sequence, wherein the first primer of the first primer pair is capable of hybridising with the first DNA sequence at a first binding site, and the second primer of the first primer pair is capable of hybridising with the second DNA sequence at a second binding site, and the second primer of the first primer pair is homologous with a section of the first DNA sequence, which homologous section of the first DNA sequence is located 5' of the site where the first primer of the first primer pair is able to bind to the first DNA sequence; - wherein the first primer of a second primer pair comprising a first primer and a second primer, is contacted with the second DNA sequence that is complementary to the first DNA sequence, and the second primer of the second primer pair is contacted with the first DNA sequence, enabling the first primer of the second primer pair to hybridise with the second DNA sequence at a third binding site, and the second primer of the second primer pair is capable of hybridising with the first DNA sequence at a fourth binding site and wherein the first primer of the second primer pair is homologous with a section of the first DNA sequence, which homologous section of the first DNA sequence is located 5' of the site where the second primer of the second primer pair is able to bind to the first DNA sequence; wherein a primer, selected from the first and second primers, is a primer comprising a primer sequence and a terminal overhang sequence at the 5' -end; and wherein, if necessary, the second primer of each primer pair at the 5' -terminus is provided with an RNA-polymerase sequence, - in a first amplification reaction (I), a DNA polymerase is used to conduct an amplification reaction using the first primer pair in the presence of suitable nucleotide compositions; - in a second amplification reaction (I), a DNA polymerase is used to conduct an amplification reaction using the second primer pair in the presence of suitable nucleotide compositions; the reaction products of the first and second amplification reaction are contacted with an RNA polymerase for transcrip- tion in order to form a first and a second ribonucleotide sequence, which ribonucleotide sequences are hybridised with each other to yield the double-stranded polyribonucleotide sequence with terminal overhang. Since the overhanging nucleotide sequence may be chosen freely, both with regard to the length and with regard to the nucleotide composition, the present invention provides an extremely flexible process for providing RNA molecules of the kind mentioned. The two amplification reactions will be performed in separate containers . In order to avoid that the description of the invention in the appended claims becomes unnecessarily complicated, it should be understood that the situations set out below, form an integral part of the present invention. There is a possibility, that the first and second sequences are not completely complementary, and it suffices that the amplified sections are adequately complementary to make possible and/or to maintain hybridisation of the polyri- bonucleotide reaction products under the conditions desired by the user. In practice, the first and second DNA sequences will be completely identical, and the DNA starting material is simply introduced into two separate containers for the performance of the amplification reactions. To each of the containers a different primer pair is added. The portion of the primers that hybridises with the first DNA sequence may be identical or different for the first and second container. This also applies for the portion of primers that hybridise with the second DNA sequence. In practice, the process according to the invention will be used mostly for preparing a polyribonucleic acid with two terminal overhangs that may or may not be complementary to each other. The invention also creates the possibility for one, and only one, of the terminal overhangs to have a length of zero ribonucleotides, which means that this is a double- stranded RNA molecule having a terminal overhang at one terminus only. The main claim would become unnecessarily complicated if the term "terminal overhang" in the present application did not also include the possibility of one of the two RNA sequences having a terminal overhang of zero length, so that in fact there is no overhang. The RNA polymerase sequence of the second primer is optional, in the sense that it is not needed if the respec- tive DNA sequence already comprises it in the desired position. In practice, the second primer will in most cases have to comprise the RNA polymerase sequence. The term "primer sequence" is understood to refer to the sequence of a primer that recognises the first or second DNA sequences, i.e. that is capable of hybridising with it so as to realise DNA polymerisation. The various steps of the process according to the invention may be executed at different locations . Although in the present application reference is made to polyribonucleic acid, no specific significance must be accorded to the term "poly", in the sense that it may also mean "oligo". Any double-stranded ribonucleic acid product is encompassed that, departing from the single-stranded reaction products, is able to hybridise or remain hybridised under the conditions desired by the user. Preferably, the double-stranded polyribonucleotide sequence is subjected to a purification step. A convenient means of doing this is gel electrophoresis . In accordance with a preferred embodiment, after transcription and preferably after hybridisation, a digestion treatment is carried out for the digestion of DNA. This facilitates the removal of DNA. As already mentioned, the double-stranded polyribo- nucleotide sequence according to the invention preferably possesses not 1 but 2 terminal overhangs. A preferred embodiment is characterized in that the terminal overhang sequence of the first primer pair is not complementary with the terminal overhang sequence of the second primer pair. In this way the termini of one double-stranded RNA molecule formed in accordance with the invention can be prevented from hybridising with each other and thereby rendering them less available for ligation to other polynucleotide molecules. In addition, by using non-complementary termini it is possible to ensure a precise orientation of the double- stranded RNA molecule in relation to another double-stranded polynucleotide molecule. The present invention makes it possible to prepare a double-stranded polyribonucleotide sequence, of which a strand has terminal overhangs both at the 3' end and at the 5' end. This interesting embodiment is characterized in that of one primer pair, both the primers possess identical or different terminal overhang sequences. The present invention also relates to a process for preparing a double-stranded polynucleotide construct. This process is characterized, in that a terminal overhang of the double-stranded polyribonucleotide sequence according to the invention is ligated to a double-stranded polynucleotide sequence, which double-stranded polynucleotide sequence possesses a complementary terminal overhang. This makes it possible to prepare RNA-RNA and RNA- DNA constructs according to choice. In this connection, it should be mentioned, that the complementary terminal overhang of the double-stranded polynucleotide sequence does not need to be completely complementary, providing that sufficient hybridisation is possible to allow ligation. Finally, the invention relates to an application of a double-stranded polyribonucleotide sequence prepared in accordance with the invention, for the transformation of a cell. It will be obvious, that the cell may be a prokary- otic or eukaryotic cell. Moreover, it may be a cell from a multi-cellular organism, such as a mammal. The present invention will now be elucidated with reference to an exemplary embodiment and the drawing, in which Fig. 1 schematically illustrates how a double- stranded RNA molecule having two 3' terminal overhangs can be prepared; Fig. 2 substantially corresponds with Fig. 1 and describes how a double-stranded RNA molecule with two 5' termi- nal overhangs can be prepared. Fig. 1 schematically shows how a double-stranded RNA molecule 16 can be prepared by implementing the process. In the example described here, the double-stranded RNA molecule has two terminal overhangs, more specifically two 3' terminal overhangs . In this example, one starts with double-stranded DNA 17, which is separated into two portions (step I) . Each por- tion is treated substantially in the same manner, and each portion provides a single-stranded RNA molecule 15', 15", which single-stranded RNA molecules 15' , 15" are hybridised, resulting in the desired double-stranded RNA molecule 16. The double-stranded DNA 17 possesses a first DNA se- quence (or strand) 1, and a second DNA sequence 2. These strands are indicated with a single or a double prime, depending on which portion they represent. Thus, in this example, the first DNA sequences 1' and 1" are identical. The same applies for the second DNA sequences 2' and 2". To prepare single-stranded RNA molecules 15, a PCR reaction (step II; PCR 1 and PCR 2) is first carried out, as described in the exemplary embodiment here by employing, for example, a Mastercycler (Eppendorf, Hamburg, Germany) . In this step, a first primer pair (primers 3, 4) and a second primer pair (primers 8, 9) are used. For a proper understanding of the term primer as used in the present application, attention is drawn to the following. For a DNA amplification an oligonucleotide sequence is needed that hybridises with DNA, and which is capable of using a DNA polymerase for chain elongation. This oligonucleotide sequence can be indicated as the actual primer. In order to prepare an RNA molecule with 3' terminal overhangs, the actual primer (shown as a black block of primers 3, 4, 8, 9) must be provided with an extra sequence 18, 19 for the primers 3, 8, respectively, and with a sequence to allow transcription (horizontally striped rectangle indicated with T7) . This latter sequence is optional for the preparation of 3' terminal overhangs, as long as the DNA nucleotide sequences provide the necessary RNA promoter sequence. If that is not the case, the primers 4, 9 are pro- vided with the RNA promoter sequences 13. In order to prevent that the two G nucleotides that are present in the T7 promoter (indicated with a horizontally striped rectangle) become incorporated in the overhang, care is taken that two C nucleotides are present at the 3' end of RNA molecule 15' . To this end two G nucleotides are present in primer 3 during PCR 1. In this way the 5' terminus of RNA molecule 15" (that for T7 RNA polymerase (step III) always starts with two Gs) is able to hybridise with the two C nucleotides of RNA molecule 15', and thus do not form part of the overhang. The second primers 4, 9 are provided with a recognition sequence 13 for T7 RNA polymerase. Obviously, different RNA polymerases and hence also different recogni- tion sequences 13 may be used for the portions. The sequences 18, 19 may be complementary, as is usually the case with double-stranded DNA obtained with restriction enzymes, but with the process according to the invention, the person skilled in the art is free to choose the length and the nucleotide composition of these sequences 18 and 19. As already mentioned, the present invention also includes the case where one (and only one) of these lengths is zero nucleotides long. After the PCR reactions using a suitable DNA poly- merase such as Pfu DNA polymerase and nucleotides, a DNA reaction product 14', 14" is obtained. By using T7 RNA polymerase and nucleotides, single-stranded RNA strands 15' , 15" are obtained. The direction of transcription is indicated with arrows . To form the desired double-stranded RNA 16 (step IV) it suffices to heat to a denaturing temperature of, for example, 65 °C. Cooling will provide the desired double-stranded RNA product. In practice it is advantageous to remove the DNA. This is easily done by using a DNase, such as DNase I. In addition, or instead of this, a purification step may be carried out using gel-electrophoresis . It is known that transcription is not a 100% reliable process. In order to improve this reliability it is therefore preferred to use prior art techniques. A particularly useful reference in this regard is Kao et al . , RNA 5_, pp. 1268-1272 (1999) . In Fig. 2 the preparation of a double-stranded RNA product 16 is illustrated, wherein the resulting RNA product 16 has two 5' terminal overhangs. The process corresponds essentially with the process described for Fig. 1. However, now the terminal overhang sequences 18, 19 are located between the primer portions actually hybridising with the DNA strands and the sequence 13 needed for T7 RNA polymerase. Within the scope of the invention, the process may be modified in various ways. For example, it is possible to prepare a double-stranded RNA molecule of which one strand has a terminal overhang at the 3' end as well as at the 5' end. To the ordinary person skilled in the art it is clear that in order for the RNA molecules prepared by the method according to the invention to be built into other double- stranded polynucleotide molecules, additional treatments such as phosphorylation and dephosphorylation may be necessary. Since a patent publication merely serves as description of an invention and not as general training to produce an expert, those who do not have the knowledge and expertise of the or- dinary person skilled in the art are referred to reference books (e.g. Molecular Cloning by Sambrook et al . ; Cold Spring Harbor Laboratory Press) and a suitable university. The double-stranded RNA molecules according to the present invention are useful for various applications. For the digestion using a Dicer enzyme in RNA interference experiments, as well as for the preparation of pharmaceutical compositions and therapies based on RNA interference, it is exactly the double-stranded RNA sequences prepared with the method according to the invention, that are more useful than RNA sequences prepared by other techniques. With regard to RNA interference, double-stranded RNA nucleotide sequences having a 3' terminal overhang are preferred.

Claims

1. A process for preparing a double-stranded polyribonucleotide sequence with terminal overhang, characterised by starting from two complementary single-stranded DNA sequences (1, 2) designated as first DNA sequence (1', 1") and 5 second DNA sequence (2',

2") - wherein the first primer (3) of a first primer pair comprising a first primer (3) and a second primer (4), is contacted with the first DNA sequence (1'), and the second primer (4) is contacted with the second DNA sequence (2)

10 that is complementary to the first DNA sequence (1'), wherein the first primer (3) of the first primer pair is capable of hybridising with the first DNA sequence (1') at a first binding site (5) , and the second primer (4) of the first primer pair is capable of hybridising with the second

15 DNA sequence (2') at a second binding site (6), and the second primer (4) of the first primer pair is homologous with a section (7) of the first DNA sequence (1'), which homologous section (7) of the first DNA sequence (1) is located 5' of the site (5) where the first primer (3) of the

20 first primer pair is able to bind to the first DNA sequence (!'); — wherein the first primer (8) of a second primer pair comprising a first primer (8) and a second primer (9), is contacted with the second DNA sequence (2") that is complemen-

25 tary to the first DNA sequence (1"), and the second primer (9) of the second primer pair is contacted with the first DNA sequence (1"), enabling the first primer (8) of the second primer pair to hybridise with the second DNA sequence (2") at a third binding site (10), and the second

30 primer (9) of the second primer pair is capable of hybridising with the first DNA sequence (1") at a fourth binding site (11) and wherein the first primer (8) of the second primer pair is homologous with a section (12) of the first DNA sequence (11'), which homologous section (12) of the

3.5 first DNA sequence (1") is located 5' of the site (11) where the second primer (9) of the second primer pair is able to bind to the first DNA sequence (1"); wherein a primer, selected from the first and second primer pairs (3, 4, 8, 9) is a primer comprising a primer sequence and a terminal overhang sequence at the 5' end; and wherein, if necessary, the second primer of each primer pair at the 5' -terminus is provided with an RNA-polymerase sequence (13) ,

- in a first amplification reaction (I) a DNA polymerase is used to conduct an amplification reaction using the first primer pair in the presence of suitable nucleotide compositions;

— in a second amplification reaction (I) a DNA polymerase is used to conduct an amplification reaction using the second primer pair in the presence of suitable nucleotide compositions; the reaction products (14', 14") of the first and second amplification reaction are contacted with an RNA polymerase for transcription in order to form a first (15') and a second (15") ribonucleotide sequence, which ribonucleotide sequences (15', 15") are hybridised with each other to yield the double-stranded polyribonucleotide sequence (16) with terminal overhang. 2. A process according to claim 1, characterised in that after the transcription with RNA polymerase, a digestion treatment is carried out for the digestion of DNA. 3. A process according to claim 1 or 2, characterised in that the terminal overhang sequence of the first primer pair is not complementary with the terminal overhang sequence of the second primer pair.

4. A process according to one of the preceding claims, characterised in that of one primer pair, both the primers may possess identical or different terminal overhang sequences.

5. A process for producing a double-stranded polynucleotide construct, characterised in that the double-stranded polyribonucleotide sequence (16) having a terminal overhang produced in accordance with one of the claims 1 to 4 is li- gated to a double-stranded polynucleotide sequence, which double-stranded polynucleotide sequence possesses a complementary terminal overhang.

6. The application of a double-stranded polyribonucleotide sequence produced in accordance with one of the claims 1 to 5, for the transformation of a cell.