DE19737442C2 - Protein-coated polyribonucleic acids, a process for their preparation and their use - Google Patents

Description

The invention relates to protein-coated polyribonucleic acids containing a natural one occurring RNA virus or RNA bacteriophage, its natural nucleic acid sequence is varied by singular or multiple insertion and / or substitution, these inserted or substituted sequences comprise 20 to 900 bases, a method for their Manufacture and its use, preferably in diagnostic procedures.

Many modern diagnostic methods today rely on the detection of a specific one Nucleic acid or on the quantification of nucleic acids. Such evidence can directly, for example by hybridizing oligonucleotide samples to one Target nucleic acid or via the amplification of nucleic acids and subsequent detection of the amplification products take place. [Bernstam, Victor A., Handbook of Gene Level Diagnostics in Clinical Practice, (1992), CRC Press, Boca Raton, USA; Congress on Advances in Nucleic Acid Amplification and Detection, June 1997, San Francisco, USA].

Evidence of viral and bacterial infections in humans and animals, evidence on Presence of pathogens and spoilage agents in raw materials and food, the Detection of translocations and mutations, the test for the identity of individuals who Proof of paternity or evidence in forensics and forensic medicine routinely today via the polymerase chain reaction (PCR) or other nucleic acid Amplification techniques (examples: polymerase chain reaction, PCR, repair chain reaction, RCR, ligase chain reaction, LCR, isothermal amplification techniques such as Strand Displacement Amplification, SDA, Transcription-based Amplification System, TAS, Self sustained sequence replication, 3SR or nucleic acid sequence-based amplification, NASBA). The presence of a specific nucleic acid is detected. [PCR: Clinical Diagnostics and Research, Eds. Rolfs, A, et al., (1992) Springer Verlag Berlin; Nonradioactive Labeling and Detection of Biomolecules, Ed. Kessler, C. (1992) Springer Berlin publishing house; Bei, A.K. et al., (1991) Crit. Rev. Biochem. Molec. Biol. 26, 301-334].  

The quantification of nucleic acids is also used in particular in Quantification of specific ribonucleic acids in cells (messenger RNA, mRNA). The Measuring the amount of mRNA transcription products can shed light on Clinical pictures or a disease status and can be used to assess effectiveness or of mechanisms of action of pharmaceuticals on patients, in cell culture and in Basic research can be used. Prominent examples are the quantity determination of cytokines (growth factors) that play a role in inflammatory processes and cancer play, or of cytochromes, which are a sensitive parameter to environmental pollution represent. [Becker-André, M., 1991, Methods Mol. Cell. Biol. 2, 189-201].

The detection or quantification of RNA is done by direct hybridization or after their transcription (reverse transcription, RT) in cDNA by means of amplification carried out. The cDNA is then quantified using a comparative method Method, that is, the detection of a known amount of nucleic acid in addition to the Target nucleic acid, the amount of which is unknown. Examples of such processes are competitive PCR and the competitive NASBA. The sequence of the nucleic acid, its amount is known to be as similar as possible to the nucleic acid sequence to be quantified is. However, it always contains sequence regions over which it or its amplification product of the can be differentiated to be quantified nucleic acid. Wear quantity standards Deletions, insertions or substitutions so that they are differential Hybridization, a nucleotide sequence analysis, a restriction fragment analysis or their Let length differentiate. [Reischl., U. and Kochanowski, B. 1995, Mol. Biotechnol. 3, 55-71].

The nucleic acids to be detected can be made from deoxyribonucleic acid (DNA) or from Ribonucleic acid (RNA) exist. For all diagnostic procedures based on the qualitative Detection or based on the quantitative analysis of nucleic acids Nucleic acid standards required for validation and for reference. DNA is very stable and can serve as a reference for diagnostic procedures. They are mostly synthetic or cloned DNA fragments of the desired sequence are used. [Lebo R.V. et al., 1990, At the. J. Hum. Genet. 47, 583-590; Mudd, J.L., et al., 1989, Crime Lab Dig. 16, 41-59; TWGDAM (Technical Working Group on DNA Analysis Methods, USA)].  

The use of appropriately produced RNA sequences is due to the lability of these Limited fabric class. RNA is extremely sensitive to degradation. You can for Example by the action of heavy and transition metal ions, by alkali and by a variety of enzymes, especially due to the ubiquitous ribonucleases be cut and degraded. RNA can therefore only take precautions, that is using ultrapure water and if possible in the presence of ribonuclease-in hibitors are handled. [Dobbeling, U. et al., 1997, BioTechniques 22, 88-90]. The RNA added to sample material is degraded very quickly. Synthetically produced RNA or RNA transcribed in vitro cannot without loss to a sample, the RNA contains degrading ingredients, are given, and is not suitable for workup and to track and assess transcription of RNA into DNA.

Part of the process of isolating the RNA from a sample and reversing its transcription works the sample in particular by enzymatic degradation and by the low efficiency of reverse transcription lost. For a direct comparison, a reference sample must be made processed together with the sample to be determined to reduce these losses determine. A synthetically or enzymatically produced reference ribonucleic acid that is not additionally protected, for example, by proteins enveloping or binding to them, is degraded even before processing begins, if it becomes an untreated one Sample material is added so that the measured values are falsified. Serves as standard today mostly a corresponding DNA sequence. A DNA standard is subject to the Digestion of the sample does not show the same degradation as the RNA sample to be determined. A DNA standard can increase the efficiency of isolation of the ribonucleic acids and the efficiency of their Do not document reverse transcription.

Ribonucleic acid standards can be chemically synthesized or by in vitro transcription getting produced. [Piatak. M. et al., 1993, BioTechniques, 14, 70-81]. RNA sequences can be replicated in vitro using the replicase from Q-beta if they correspond Carrying recognition sequences from the genome of the phage Q-beta. [Lizardi et al., 1988, Biotechnology, 6, 1197-1202]. RNA sequences can be used as a quantitative standard for a RT-PCR can be used. [Fille et al., BioTechniques 23 (1997) 34-36)]. You are not against protected against degradation.  

Standards for ribonucleic acids can also consist of the material to be detected. These can occur in the case of human pathogenic viruses, for example when using a Blood or serum sample can be infectious, fall under epidemic regulations and laws and therefore be limited in their handling. Standards are made from a natural isolate remained silent on a large scale. Blood samples are not homogeneous and aliquots blood or serum can contain very different virus titers. Natural isolates cannot be used as a competitive standard as they are not mandatory Contain sequence variations that make them distinguishable.

There have been retroviral packaging systems based on human and monkey viruses developed the packaging of ribonucleic acids with sequence variations in Virus particles allow, for example, the effect of certain gene sequences research or in the future certain RNA sequences for gene therapy in cells introduce. These systems are based on virus particles similar to the AIDS virus. The Virus particles are produced in cell culture from which novel infectious viruses are made can develop. [Flamant, F., Cosset, F.-L. and Samarut, J., J. Mol. Med. 73 (1995) 181-187]. Constructs have also been reported that were genetically unstable and that have lost varied sequences in whole or in part. [Olsen et al., J.Virol. 64 (1990) 5475-5484). The production of varied polyribonucleic acids in viruses is relatively complex, dangerous and requires handling cell cultures. The preparations contain minor Numbers of genome equivalents (10E5 to over 10E7 genome equivalents per ml) to include them in to manufacture a very large amount. [Cosset et al., J. Virol. 69 (1995) 7430-7436].

There are a large number of RNA phages in bacteria that are divided into several families. she consist of a polyribonucleic acid that is derived from a large number of protein molecules, the so-called "coat protein" is coated. The phage nucleic acid encodes a polymerase, your Coat protein and other protein factors. The phages are also host factors dependent. Some phages can only infect "male" bacteria, that is bacteria with an F-particle (F +). The phage Q-beta encodes a "maturation" protein a2, a "coat" Protein, a "readthrough" protein a1, which as a result of a readout event via a Stop codon represents a C-terminally extended "coat" protein, and an RNA polymerase so-called replicase. The "readthrough" protein is at the host infection by the Q-beta  Phages involved. [Weissmann, C., FEBS Letters 40 (1974) S10-S18]. It is known that a Failure of each individual protein as a result of variations in the respective coding Nucleic acid sequences in the case of the phage Q-beta can be complemented by the Affected protein expressed in trans, that is, elsewhere in the host organism becomes. [Priano C. et al., J. Mol. Biol. 249 (1995) 283-297].

The phage genome forms a distinctive secondary structure. Changes in the genome of the Phages are usually not genetically stable and become within a few pha gene generations reversed or neutralized by further changes in the genome. A stable folding pattern is obviously created again. [Arora R. et al., J. Mol. Biol. 258 (1996) 433-446]. Corresponding results have been obtained with the phage M2 been [Ref. 196054h]. RNA phages cannot easily be varied in a genetically stable manner.

RNA phages have been used to produce recombinant proteins or peptides. The coat protein from phage Q-beta has been artificially extended [Kozlovska, T. M. et al., Intervirology 39 (1996) 9-15; Ref. 49884b]. The "coat" protein forms extremely stable particles out. The "coat" proteins are connected to each other by disulfide bridges. [Golmohammadi, R. et al., Structure 4 (1996) 543-554]. Modifications are also made to the read-through protein a1 from phage Q-beta [Ref. 44289, 85295] and on proteins of phage R17 [Ref. 9349g] Have been carried out.

It has now been found that polyribonucleic acids with a freely definable sequence can produce portions that contain a desired sequence, and that of a protein are covered, which protects them against degradation. With these protected Polyribo For example, nucleic acids can be used to detect the detection limits of qualitative nucleic acids check wisely; it can also be used to produce standards which, after being added to the determining sample to be degraded to the same extent as the substance to be determined and can be used as a quantity standard in this way. You allow, for example show the competitive quantification of RNA directly from the sample material without the above to have to accept the disadvantages mentioned.  

The present invention therefore relates to protein-coated polyribonucleic acids which characterized in that they contain a viral RNA or bacteriophage RNA, wherein the natural nucleic acid sequence by singular or multiple insertions and / or Substitutions is varied, these inserted or substituted sequences 20 to 900 Include bases.

The newly introduced freely definable sequence sections consist of synthetic Sequences or from possibly varied genome sequences of other organisms. The related genome sequences are preferably those of diagnostic relevance, that is, that precisely these sequence sections are used in diagnostic procedures. With these Sequence sections are, for example, the binding sites for PCR primers or around binding sites for oligonucleotide probes.

In particular, the genome sequences which may be varied are or Sections around those from human pathogenic RNA viruses, for example retro and flaviviruses. Sequences from the HIV, HCV, HGV, influenza, picorna, yellow fever, hanta or dengue viruses.

Furthermore, the genome sequences which may be varied are or Sections around coding sequences from mammals, for example sections from the Messenger RNA from growth factors (cytokines), their cellular amount in diagnostic Procedure is determined.

The synthetic sequences can be functional ribonucleic acids for Example of ribozymes or aptamers.

In a preferred embodiment, the protein-coated polyribonucleic acids are made from the RNA bacteriophage Q-beta its nucleic acid sequence as described above is varied.

The variation of the nucleic acid sequence preferably leads to the genome sequence being so far changed is that the underlying bacteriophage or virus is no longer without Add additional elements independently.  

Sequences are preferably substituted, that is, the total length of the sequence is not or varies only slightly. The newly introduced freely definable sequence sections have approximately the same length as the sequences deleted from the virus or phage genome.

In the inserted sequence variation, genomic regions of the phage are selected that either no functional area for the phage or only the reading frame for a single one Concern protein. The sequence variation is preferably directed to a genome region of the Limit phages that encode exactly one protein that is required for the multiplication of the phage is necessary. As a result, phages that carry the desired sequence variation cannot multiply independently if the missing factor is not supplied by the host cells becomes. To produce the protein-coated polyribonucleic acid according to the invention Phages are thus used bacterial cells that provide this missing factor. This factor consists of the unchanged coding gene for the protein, the Reading frame is affected by the sequence variation. The gene can be integrated into the host genome may be or may be on a separate genetic introduced into the host organism Element.

Bacterial cells, which are not, are preferably used to produce such a phage can be infected by a phage (genetic marker F⁻). This allows the Do not multiply phages on their own and mutations through multiple passages of the Phages are excluded. The genome of an RNA phage is under a high one Selection pressure.

In a particularly preferred embodiment, the protein-coated poly ribonucleic acids from the bacteriophage Q-beta, in the nucleic acid sequence of which 3 'region of the gene for the "read-through protein" a1 in whole or in part by others, free determinable sequences is replaced. This sequence variation will preferably approximate Exchanged sequence sections of the same length. Insertions are preferred in places inserted, which have little effect on the folding structure of the RNA phage genome. Such Places are, for example, the ends of stem loops. The 5 'terminal area for that "Readthrough Protein" a1 codes for the "Coat" protein. The reading frame for the "Coat" protein  is preferably terminated with a constitutive stop codon [TGA] and otherwise not varied.

The present invention also relates to a process for the preparation of the protein-coated polyribonucleic acids described above, which is characterized in that:

• (a) In an RNA phage genome or RNA viral genome that acts as a DNA sequence is cloned, by insertion or substitution of 20 to 900 bases a foreign, inserts freely definable nucleic acid sequence and thus the genome varies and
• (b) the varied genome sequence into a suitable host cell by known methods brings in which viruses or phages are produced, which the desired contain varied polyribonucleic acid packed in a protein envelope.

One goes for the production of the protein-coated polyribonuclein according to the invention acid from an RNA phage genome or from an RNA viral genome as a vector, which as DNA sequence is cloned.

The natural genome is changed by insertion or substitution. Such a variation almost inevitably leads to a failure of essential functions for the virus or the Phages. The insertion is therefore made at a position where there is no functional area of the Phage is affected or at least only one function is affected, that is, preferably at a point that destroys the coding region for only one protein. Of the Loss of function is complemented by providing this gene in the host cell.

The varied genome sequence is brought into host cells in which it is used as a template for Production of the varied ribonucleic acid is used. The host cells produce virus or Phage particles that contain the desired polyribonucleic acid.

The following description is intended to explain the method according to the invention in detail without it limit to this.  

A Use of vectors in host cells

A is used for the production of the protein-coated polyribonucleic acid according to the invention RNA virus or RNA phage used as a vector. With these vectors it can become Examples are bacteriophages, retroviruses, influenza viruses or plant viruses that act as Genome contain single-stranded or double-stranded RNA. The production of the proteinum enveloped ribonucleic acid takes place in the respective natural or in a compatible Host cell of the virus or phage used as a vector.

B Origin, properties and handling of the freely definable sequence B1 Origin of the freely definable sequence

The freely definable sequence that is to be brought into the vector is either synthetically produced or isolated from another organism and optionally varied. It is available as a DNA sequence. This can be a DNA fragment or a cloned DNA. It has a length of 20 to 900 bases.

B2 Properties of the freely definable sequence

To insert the freely definable sequence into the vector, use for example the targeted cloning via restriction sites. The selected interfaces are preferably singular, that is, they do not exist either in the vector or in the freely definable one Sequence elsewhere.

B3 Add the interfaces to the freely definable sequence

If the freely definable sequence does not contain any suitable restriction sites, these become added to the sequence. You can use directed mutagenesis methods or by means of Polymerase chain reaction can be artificially added or inserted by using the freely determinable Sequence is amplified with primers with their 3 'regions complementary to this Are sequence and carry the interfaces as an addition at their 5 'ends.

C production of vectors C1 Isolation of genome sequences

The ribonucleic acid genome of an RNA virus or an RNA bacteriophage is isolated and rewritten in "Copy DNA" (cDNA) (reverse transcribed).

C2 cloning of genome sequences

The cDNA obtained is cloned, for example, in E. coli. This makes the genomic Sequence accessible for genetic engineering manipulations. May be used for cloning Plasmids used to transfer the cloned genome sequences into their original Lichen or allow a compatible host organism.

C3 expression of genome sequences

The cloned genomic sequence is placed behind regulatory elements, the one Allow transcription of this sequence in the host organism. It will be preferred regulatory elements that can be induced, that is, elements that are used allow experimental regulation of expression. If the genomic sequence in When the host organism is transcribed, the ribonucleic acid genome and the one used are created RNA virus or RNA bacteriophage is expressed.

C4 properties of virus or phage genome sequences

When manipulating a viral genome or a phage genome, almost inevitably functional sequence areas changed. You can use the coding sequence for Proteins, regulatory areas on the nucleic acid, areas required for replication are important or other functions essential for the virus or for the phage affect. Some sequence areas have different functions at the same time involved. A variation in the total length of the genomic sequence can result in replication and affect the packaging.

D properties of a variation in genome sequences D1 Length of the varied sequence

When manipulating the genomic sequence, areas are preferably substituted instead of inserted so that the overall length of the genomic sequence is little affected.  

D2 Complementation of a functional failure

The failure of certain functions can be complemented. Areas in a Pha gene or in a viral genome that code for only one protein can be destroyed, replaced or be deleted so that the varied phage or virus genome is no longer a template for the error-free expression of this protein can serve. If this protein is within the Host cell can be produced regardless of the varied phage or virus genome, the loss of function otherwise associated with the loss of this protein is complemented.

D3 Selection of the region to be varied in the genome

A variation in the genome of the phage or virus is preferably found in only one Protein coding region takes place without further, for example, regulatory elements to destroy. For example, the variations are preferably made in places at which influence the folding pattern of the genomic RNA as little as possible.

D4 Properties of genomes with dysfunctions

From a cloned genome that carries a variation that leads to the failure of an essential one Function for the virus or for the phage leads, no functional virus or phage can arise. The failure of a certain function prevents the propagation of the affected Phages or virus in host cells that are not an additional compensating factor produce. Cloned genomes with such a variation are safe against an uncontrolled one Spread of a virus or phage. From the protein-coated ribonucleic acid, the natural sequence is varied, no functional virus or phage can be replicated.

E variation of a genome sequence by insertion or substitution E1 Selection of the location for a variation in the genome

Locations or areas that do not exist are preferably selected for the variation in the genome Cause loss of function for the phage or in those by insertion or by Substitution affects only the function of a protein coding gene. If not Loss of function, there is no need for a complementation system. However, it is preferred to select a location or an area for variation that exactly one Reading frame for a protein is destroyed, but is not otherwise functionally important. Failure  this reading frame is replaced by a copy on another genetic element Example of a plasmid, complemented. The malfunction has the advantage that the such a varied phage or virus cannot reproduce and spread without this element.

E2 Incorporation of restriction sites into a cloned virus or phage genome

Freely determinable sequences are preferably inserted by cloning via Restriction interfaces. To do this, the genomic sequence is cloned to appropriate ones Make interfaces for restriction enzymes inserted using genetic engineering methods using the restriction ends of the freely definable sequence to be inserted are compatible. The These interfaces can be inserted, for example, via directed mutagenesis or via the Polymerase chain reaction take place. When using the latter, the entire cloned Genome sequence amplified with primers that target the desired restriction sites wear their 5 'ends. The primers bind in the genomic sequence; by choosing the Part of the genomic sequence can be deleted. During amplification creates a linear copy of the entire sequence, the deletions in the target area may contain genomic sequence. The PCR amplification product can be used after a Restriction digestion can be used directly to insert a freely definable sequence. The Amplification product can be circularized and cloned to serve as a template for insertion to be used by new sequence sections.

E3 insertion or substitution with a freely definable sequence

The freely definable sequence is inserted into the cloned genomic sequence. she will inserts or substitutes an area within the genomic sequence. This Area is due to the positioning of the restriction interfaces used for this given. The freely definable sequence provided with restriction sites and the one with Restriction sites modified genomic sequence with the respective Restriction enzymes digested and ligated together. The ligation products are cloned. The clones are checked to ensure that they correspond to the desired sequence.

F Complementation of functional failures F1 complementation by producing a protein product in the host cell

The host cells for the phages or for the viruses are equipped with the affected gene, which has become inoperative in manipulating the genomic sequence to the protein manufacture and complement the associated functional failure. The Provision of the coding sequence for the host cell can be done either by insertion this gene sequence into their genome or by inserting a vector. This complementing sequence can also be contained on the same vector that the varied carries genomic sequence.

F2 Isolation of the gene for the complementing gene product

The gene for this protein product is derived from the phage genome or from the virus genome isolated and cloned in bacteria.

F3 expression of a complementing gene in the host cell

The complementing gene is under the control of a suitable regulatory element placed and brought into the host cell by means of a suitable plasmid or vector. The regulatory element controls the permanent or inducible expression of the gene. As a result, the host cell has the corresponding protein to make it functional Loss generated by the variation in the cloned genomic sequence increases complement. A suitable plasmid or vector is with the simultaneous Presence of the vector compatible in the same host cell. For bacteria to do this for example the origins of replication (ori) of the plasmids used with one another be compatible. The vectors must have different selectable resistance markers.

G Production of protein-coated polyribonucleic acid G1 Introduction of the varied gene sequence into the host cells

There are those belonging to the respective system, ie the RNA viruses or RNA phages or compatible host cells are used. The host cells express the gene that is produced by the Variation of the cloned genome sequence caused loss of function complemented (F3). The cloned gene sequence that carries the desired variations (E3) is in the host cells brought.  

G2 Expression of phages or virus particles

The host cells that contain the varied gene sequence and the complementing sequence express constitutively or after induction phage or virus particles, which instead of a Pha gene or virus genome carry a varied genome that contains the freely definable sequence.

G3 Isolation of phages or virus particles

Depending on the type of system used, the phages or virus particles generated are transferred to the same way as the naturally occurring phages or viruses from the medium or after Lysis of the cells isolated from them by centrifugation, precipitation or extraction. The phages or virus particles can also with the surrounding host cells, if necessary be deactivated, used.

H methods

Methods for manipulating nucleic acids and introducing them into heterologous Systems can be found in the "Molecular Cloning" manual [Sambrock, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning. A Laboratory Manual (1996) Cold Spring Harbor Press, USA].

Another object of the invention is the use of the invention protein-coated polyribonucleic acids, in particular in diagnostic methods, for Example for the qualitative and quantitative detection of ribonucleic acids Positive control of the detection of virus RNA, to control the efficiency of procedures for the purification and isolation of ribonucleic acids or to control the efficiency of the Reverse transcription of ribonucleic acids.

The different possible uses are explained in more detail below, without restrict the uses to these examples:

A use as a sequence-specific standard for diagnostic and other methods, where the presence of a specific ribonucleic acid is detected

In procedures where the presence of a particular ribonucleic acid in a sample is detected, the protein-coated polyribonucleic acids according to the invention as Standard used to determine the suitability of the procedure used document or check or to limit the detection of the method used determine. The protein-coated polyribonucleic acid used as standard can be in their freely definable part of the sequence of the ribonucleic acid sequence to be detected entirely or in parts.

Method of testing for the presence of a particular ribonucleic acid in a sample consist of sample preparation and isolation of ribonucleic acid and its detection. Working up is carried out by extraction, for example using phenol / chloro Form extraction, by enzymatic digestion, for example by means of proteinase, by treatment with detergents or with amphiphilic substances, by treatment with denaturing agents Salting, by heating, by frequent freezing and thawing or by mechanical Information or a combination of these methods. The further enrichment of the nucleic acid can be bound to a solid phase, for example to anion exchangers, silica gel or membranes or on glass or by extraction. Evidence is provided by direct hybridization of a suitably labeled oligonucleotide probe to the target nucleic acid or after transcription into DNA, amplification of a specific DNA fragment and Detection of this fragment via hybridization, determination of the length, restriction digestion or DNA sequencing.

The protein-coated polyribonucleic acids according to the invention become the sample material added and processed with the sample or in a separate batch analyzed.

B Use as an internal standard for diagnostic and other procedures in which the Presence of a specific ribonucleic acid is detected

In procedures where the presence of a particular ribonucleic acid in a sample is detected, the protein-coated polyribonucleic acids according to the invention as Standard used to determine the suitability of the procedure used document or check or to limit the detection of the method used  determine. The protein-coated polyribonucleic acid used as standard has one similar sequence to that to be detected or is a variation of this sequence and accompanied all steps in the diagnostic process to replace them instead of the sequence to be detected, or in a subset separated from the sample to be determined, or afterwards, if the sequence to be detected was not ascertainable.

C Use as a comparative quantity standard for the quantification of RNA virus nucleic acid and messenger RNA (mRNA)

In procedures where the amount of a particular ribonucleic acid in a sample is detected, the protein-coated polyribonucleic acids according to the invention as Standard can be used to serve as a quantity reference and for calibration curves for Generate methods to determine the quantities of unknown samples can. The protein-coated polyribonucleic acid used as standard can be free in its determinable part of the sequence of the ribonucleic acid sequence to be detected in whole or in Parts.

D Use as a competitive quantity standard

In procedures where the amount of a particular ribonucleic acid in a sample is over If competitive processes are detected, the protein-coated according to the invention can Polyribonucleic acids are used as standard to work as a competitor sequence. The protein-coated polyribonucleic acid used as standard corresponds in its free determinable part of the sequence of the ribonucleic acid sequence to be detected. The standard comes in different quantities with the unknown sample to be determined processed and analyzed. The quantity is determined by competitive methods.

E Use to control the enrichment and isolation of ribonucleic acids

In methods in which ribonucleic acids are isolated, the inventive Protein-coated polyribonucleic acids are used as the standard for fitness to document the procedure used as an accompanying sample. The one used Nucleic acid sequence is not identical to the sequence of the nucleic acid to be detected.  

F Use as a standard to check the detection capability of laboratories

In procedures where the presence or amount of a particular ribonucleic acid is detected in a sample, the protein-coated according to the invention Polyribonucleic acids are used as standard to demonstrate the detection ability of a Laboratory, institution or person or procedure.

G Use to determine the efficiency of methods for isolating RNA

In methods in which ribonucleic acids are isolated, the inventive Protein-coated polyribonucleic acids are used as the standard for fitness and document the efficiency of the procedure used. The one used as standard protein-encased polyribonucleic acid can be used in its freely definable part of the sequence of the Correspond to the ribonucleic acid sequence to be detected in whole or in part.

H Use to control reverse transcription of RNA

In processes in which ribonucleic acids are reverse transcribed, the protein-coated polyribonucleic acids according to the invention are used as standard, to document the suitability and efficiency of the procedure used.

I Use as a control reagent

For reagents and laboratory kits for the isolation of ribonucleic acids and their reversers The protein-coated polyribonucleic acids according to the invention can be transcribed into DNA can be used as standard and function control.

J Use to control cleaning and disinfection processes

The protein-coated polyribonucleic acids according to the invention can be used as a sample to improve the efficiency of cleaning and disinfection measures Check inactivation of RNA viruses.  

K Use for labeling samples

The protein-coated polyribonucleic acids of the invention can be used to: To mark samples of all kinds. The freely definable part in the sequence can be used for Coding of origin, type or other information can be used and later using a appropriate diagnostic procedures are analyzed and "read".

L Use as a sample for hybridization

The protein-coated polyribonucleic acids according to the invention can be prepared to to use the RNA isolated therefrom as hybridization samples. The RNA is up to theirs Preparation preserved by its protective protein coat. The freely definable sequence contains the areas used for hybridization with a complementary sequence become. The polyribonucleic acids according to the invention can, for example, be labeled Contain nucleotides that are built into the host organism and used for their detection can be used. Hybridized polyribonucleic acids, the variations of a Phage genomes can also be represented by in vitro replication using the RNA for this specific RNA-dependent RNA polymerase replicase can be detected.

M Use as a binding sample

The protein-coated polyribonucleic acids of the invention can be used to: To produce RNA for binding purposes. The RNA is up to its isolation by its protective protein coat preserved. The freely definable sequence contains areas that can bind certain structures (aptamers). The polyribonucleic acids according to the invention can contain, for example, labeled nucleotides that are built into the host organism and which can be used to prove them. Polyribonucleic acids, the one Binding structure and representing the variations of a phage genome can also by in vitro Replication by means of the RNA-dependent RNA polymerase specific for this RNA Replicase or detected by RT-PCR amplification.

O Use as a functional ribonucleic acid

The protein-coated polyribonucleic acids of the invention can be used to: to produce functional ribonucleic acids, for example ribozymes. The sequences are up preserved for their use by their protective protein sheath. The freely definable Sequence contains areas that contain functional sequences.

The following examples illustrate the feasibility of the present invention without this restrict to the examples:

example 1 Introduction of interfaces into a cloned Q-beta phage genome

The plasmid pBRT7Qβ was developed by Prof. Dr. Hans Weber, Institute of Molecular Biology, University of Zurich, provided [published in Barrera I., et al., J. Mol. Biol. 232 (1993) 512-521]. The plasmid carries the phage Q-beta genome behind a T7 promoter, a β-lactamase gene (ampicillin resistance) and the ColE1 origin of replication. This Plasmid leads to the production of Q-beta phages in transformed Escherichia coli (E. coli), that can produce the T7 RNA polymerase. The entire nucleotide sequence from this Plasmid is disclosed as sequence 1.

The plasmid pBRT7Qβ is transformed into a PCR product using two oligonucleotide primers which comprises the entire plasmid and the ends of which are in the gene for the "readthrough" Protein a1. The primers have restriction sites at their 5 'ends. By Religation of this product creates a plasmid with the Q-beta phage sequence and one Variation in al gene.

100 ng plasmid pBRT7Qβ with 60 pmol each of the oligonucleotide primers [82593], 5'-ATT CTA GAG CCG TGG CAA TGG TTA TAT TGA CCT TGA TGC G (Sequence 2) and [82594], 5'-TAT CTA GAA AGC TTA ATA CGCTGG GTT CAG CTG ATC AAT AGC ATC G (sequence 3), with 1 µl 10 mM deoxyribonucleotides (dNTPs), 10 mM Tris-HCl, pH 8.3, 2 mM magnesium chloride, with 2 u Taq DNA polymerase (Hoffmann La-Roche) and 0.2 u Pwo DNA polymerase (Boehringer Mannheim) in a volume of 100 ul  amplified. The reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 55 ° C and 2 min at 72 ° C and finally 20 min at 72 ° C (Perkin Elmer GeneAmp 2400). The Amplification product is with 50 ul phenol (Roth) and with chloroform / isoamyl alcohol (25: 1) extracted. 10 μl of 5 M potassium acetate and 350 μl of ethanol are added to the aqueous phase. The precipitated DNA is pelleted by centrifugation. The pellet is made with 70% ethanol washed and dried in vacuo. The DNA is dissolved in 20 µl water and with the Restriction enzyme Hind III (Boehringer Mannheim) according to the manufacturer's instructions cut (2 hours at 37 ° C). The reaction mixture is made up to 100 µl with water and extracted with phenol as above and precipitated with ethanol. The DNA pellet is in 20 ul Dissolved water and with 1 u T4 DNA ligase (Boehringer Mannheim) according to the instructions of the Manufacturer ligated (16 hours at room temperature). Electrocompetent E. coli cells (DH5alpha) are transformed with 0.5 µl of the ligation mixture. The colonies obtained are picked and dressed. Plasmid DNA is isolated from overnight cultures (Nucleobond Kit, Machery-Nagel, according to the manufacturer's instructions). The plasmid DNA is controlled by restriction digestion and DNA sequencing (Replicon Sequencing service, Berlin).

The sequence of the plasmid pBRT7Qβd is disclosed as sequence 4. The plasmid contains a varied Q-beta phage genome, whose reading frame for the "coat" protein with a TAA stop Codon is complete. The gene for the read-through protein carries a deletion of 264 Base pairs and two different singular restriction sites Hind III (AAGCTT) and Xba I (TCTAGA) at this point.

Example 2 Preparation of a plasmid with a short sequence section from the human Immunodeficiency virus (HIV) genome

The plasmid pBRT7Qβd is mixed with the restriction enzyme Hind III (Boehringer Mannheim, linearized according to the manufacturer) and dephosphorylated with alkaline phosphatase (Boehringer Mannheim, according to the manufacturer), extracted with phenol and with ethanol precipitated. The linearized plasmid is made with the two 5'-phosphorylated synthetic Oligonucleotides [81885], 5'-Ph-AGC TCA GTG GGG GGA CAT CAA GCA GCC ATG CAA AT (Ph = phosphate, sequence 5) and [81886], 5'-Ph-AGC TAT TTG CAT GGC TGC  TTG ATG TCC CCC CAC TG, (sequence 6) ligated with T4 DNA ligase (Boehringer Mannheim, according to the manufacturer). Become competent E. coli cells DH5alpha transformed. The colonies obtained are picked and dressed. Out Plasmid DNA is isolated overnight cultures (Nucleobond Kit, Machery-Nagel, according to Manufacturer's instructions). The plasmid DNA is via a restriction digest and DNA Sequencing checked.

The sequence of the plasmid pBRT7QβSK145 is disclosed as sequence 7.

Example 3 Generation of a plasmid with two sequence sections from the HIV genome

Using the two synthetic oligonucleotides [82614] 5'-TAA AGC TTA GTG GGG GGA CAT CAA GCA GCC ATG CAA ATA CCC GAT CCG GTT ATT C (Sequence 8) and [82615] 5'-TTC TAG ATG CTA TGT CAG TTC CCC TTG GTT CTC TAT CCC AAT CAC GCC ACT (Sequence 9) is amplified by PCR using a 286 DNA fragment. As The Q-beta phage genome on the plasmid pBRT7Qβ serves as a template. The reaction solution contains 100 µM deoxyribonucleotides (dNTPs), 50 µM both oligonucleotide primers in 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and with 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 µl. The reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400). The sequence of the product is given as sequence 10. The product comes with phenol extracted and precipitated. The product is with the restriction enzymes Xba I and Hind III digested (Boehringer Mannheim, according to the manufacturer), extracted with phenol and like.

The plasmid pBRT7Qβd with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and extracted with ethanol precipitated.

The cut plasmid and the cut amplification product are combined ligated, E. coli are transformed and plasmid DNA is prepared and by DNA Sequencing verified.  

The sequence of the plasmid pBRT7QβSK145-431 is disclosed as sequence 11. The plasmid contains the sequences SK145 5'-AgT ggg ggg ACA TCA AgC AgC CAT gCA AAT (sequence 12) and SK431 5'-TgC TAT gTC AgT TCC CCT Tgg TTC TCT (sequence 13) in one Spacing of 214 base pairs; the fragment has the sequences SK145 and SK431 a length of 271 base pairs.

Example 4 Production of a plasmid with a long section from the HIV genome

The polymerase chain reaction turns the HIV I genome into a fragment Restriction sites amplified. The PCR is carried out with the oligonucleotides [84673] 5'-ATT AAA gCT TAg Tgg ggg gAC ATC AAg C (sequence 14) and [84675] 5'-TA TCT AgA TgC TAT gTC AgT TCC CTT Tg (sequence 15) and a cDNA template from HIV I carried out. The reaction solution contains 100 µM deoxyribonucleotides (dNTPs), 50 µM both oligonucleotide primers, 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 µl. The reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400). The reaction product is with the restriction enzymes Hind III and Xba I (Boehringer Mannheim, according to the Manufacturer) cut extracted with phenol and precipitated with ethanol.

The plasmid pBRT7Qβd with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and extracted with ethanol precipitated.

The cut plasmid and the cut amplification product are combined ligated, E. coli are transformed. Plasmid DNA is prepared and by DNA Sequencing verified.

The sequence of the plasmid pBRT7QβSK145-102-431 is disclosed as sequence 16. It contains a 142 base pair (sequence 17) long sequence section from HIV I.  

Example 5 Production of a plasmid with a long section from the hepatitis virus C (HCV) Genome

The polymerase chain reaction turns the HCV genome into a fragment Restriction sites amplified. The PCR is carried out with the oligonucleotides [84672] 5'-ATT AAA gCT TgC AgA AAg CgT CTA gCC (Sequence 18) and [84671] 5'-TAC TCTAgA CTC gCA AgC ACC CTA TC (sequence 19) and a cDNA template from HCV guided. The reaction solution contains 100 µM deoxyribonucleotides (dNTPs), 50 µM both Oligonucleotide primer in 10 mM Tris-HCl, pH 8.3 with 2 mM magnesium chloride and with 2 u Taq DNA polymerase (Hoffmann La-Roche) in a volume of 50 µl. The reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400). The reaction product is with the restriction enzymes Hind III and Xba I (Boehringer Mannheim, according to the Manufacturer) cut extracted with phenol and precipitated with ethanol.

The plasmid pBRT7Qβd with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and extracted with ethanol precipitated.

The cut plasmid and the cut amplification product are combined ligated, E. coli are transformed. Plasmid DNA is prepared and by DNA Sequencing verified.

The sequence of the plasmid pBRT7QβHCV is disclosed as sequence 20. It contains a 216 Base pair (sequence 21) long sequence section from the HCV genome.

Example 6 Production of phage particles

The plasmid pP + QβRT was developed by Dr. Donald R. Mills, Dept. Microbiology, State University of New York, USA. It is 8.1 kb in size and carries the pSM1  Origin of replication (ori) compatible with the ColE1 ori from the pBRT7Qβ plasmid. The plasmid carries a gene for resistance to trimethoprim and the gene for the "Readthrough" protein a1 under the control of the P + promoter. This plasmid leads to one constitutive expression of al protein in transformed E. coli. [Arora et al., J. Mol. Biol. (1996) 258, 433-446].

Competent E. coli from strain BL21 (DE3) (Stratagene) or BL21 (DE3) are included nP + QβRT and an nBRT7Qβ plasmid (e.g. nBRT7QβSK145). The clones are selected for ampicillin and trimethoprim resistance (LB agar with 75 mg / l Ampicillin, sigma, 50 mg / l trimethoprim, sigma). The cells are in one overnight Pre-culture grown (LB medium with ampicillin and trimethoprim, 37 ° C) and in one Dilution of 1:25 freshly inoculated. The cultures are added after 105 min by adding 5 g / l isopropylthiogalactoside (IPTG, Sigma) induced and shaken at 37 ° C for a further 3 hours. The culture brightens up through the lysis of cells. The culture is cooled to 4 ° C and with 1/100 volume mixed with chloroform. By centrifugation (10 min, 3500 rpm) The supernatant obtained is used directly or by precipitation with polyethylene glycol concentrated. The phage supernatant is made up with RNase A (5 g / l, Sigma) and DNase I (1 g / l, Boehringer Mannheim) treated.

Example 7 Preparation of ribonucleic acid from phage particles

100 µl phage supernatant are mixed twice with 50 µl phenol (Roth) and with Chloroform / isoamyl alcohol (25: 1) extracted. The aqueous supernatant is treated with DNase I (Boehringer Mannheim) treated (1 g / l, 30 min at room temperature). The solution will be like extracted with phenol above and precipitated with ethanol (0.5 M LiCl, 3 volumes of ethanol) and through Centrifugation pelleted. The pellet is washed with 70% ethanol and extracted. The Ribonucleic acid is obtained in 1 ml TE buffer (10 mM Tris-HCl, pH 7.5; 1mM EDTA) resolved and determined photometrically.

Example 8 Analysis with the polymerase chain reaction (PCR) and RT-PCR

Ribonucleic acid according to Example 7 is used as a template. For the template RNA from the plasmid pBRT7QβSK145-431 (sequence 11) and pBRT7QβSK145-102-431 (sequence 16) the oligonucleotide primers SK145 (sequence 12) and SK431 (sequence 13) or for pBRT7QβSK145-102-431 the combination GAG3, 5'-ATCAATgAggAAgCTgCAgAATgggg (Sequence 22) and SK431 used. For RNA from plasmid pBRT7QβHCV (sequence 20) the oligonucleotide primers HC3, 5'-gAAAgCgTCTAgCCATggC (sequence 23) and HC4, 5-CCACAAggCCTTTCgCgACC (sequence 24) used for the reactions.

The PCR amplification is carried out in a volume of 50 μl with 100 μM deoxyribonucleotides (dNTPs), 50 µM of both oligonucleotide primers in 10 mM Tris-HCl, pH 8.3 with 2 mM Magnesium chloride and performed with 2 u Taq DNA polymerase (Hoffmann La-Roche). As 5 μl of RNA solution from Example 7 are used in each case. The reaction conditions are 35 cycles with 1 min at 95 ° C, 1 min at 60 ° C and 1 min at 72 ° C and finally 10 min at 72 ° C (Perkin Elmer GeneAmp 2400).

No amplification product results in any reaction batch.

The same templates (10 ul) are in the presence of both oligonucleotide primers with M-MuLV Reverse transcriptase (peqlab, according to the manufacturer) rewritten in DNA. Of the half the batch is used in the PCR as above. When using RNA from that Plasmid pBRT7QβSK145-431 and primers SK145 and SK431 form a 271 bp Fragment using RNA from the plasmid pBRT7QβSK145-102-431 and the Primers SK145 and SK431 create a 142 bp fragment when using RNA A 99 is produced from the plasmid pBRT7QβSK145-102-431 and the primers GAG3 and SK431 bp large fragment, when using RNA from the plasmid pBRT7QβHCV and the Primers HC3 and HC4 result in a 216 bp fragment of double-stranded DNA which is in the Agarose gel can be detected.

Example 9 Quantification of ribonucleic acid

Starting from the plasmid pBRT7QβSK145-431 (sequence 11) according to Example 7 Ribonucleic acid obtained from 100 ul phage supernatant. The product is sold in increments of Diluted 1:10 in TE buffer from 10E-1 to 10E-9. In each case, 10 μl are added according to Example 8 Reverse transcriptase and PCR amplification with the oligonucleotide primers SK145 and SK431 treated. The PCR amplification is carried out in five batches. The Reactions of the dilutions up to 10E-6 result in an amplification product, the dilution 10E-7 produces a product in two out of three approaches. From this series of dilutions follows a number of about 2 x 10E9 genome equivalents ml of phage supernatant.

Example 10 Analysis of a blood sample for HIV infection

1 ml of whole blood from a non-HIV-infected blood donor is diluted with 10 µl of a 10E-6 Sample of a phage supernatant, obtained using the plasmid pBRT7QβSK145-431, transferred. The ribonucleic acid is isolated with an isolation kit RNAeasy (Qiagen, according to from the manufacturer) isolated and analyzed with RT-PCR as described in Example 8. For the For amplification, the SK145 and SK431 oligonucleotide primers are used. The analysis shows a PCR product with a length of 271 bp.

Example 11 Determination of a virus by competitive quantification

1 ml of whole blood from a non-HIV-infected blood donor is diluted with 12.5 µl of a 10E-6 The phage supernatant from pBRT7QβSK145-102-431 was added, corresponding to about 25,000 Genome copies of HIV. The mixed blood sample is divided into five aliquots A, B, C, D and E. separated, each containing 5000 genome equivalents, and each with 10 ul Phage supernatant from pBRT7QβSK145-431 with dilutions of 10E-8, 10E-7, 2 × 10E-7, 10E-6 and 2 × 10E-5 offset, corresponding to 200, 2000, 4000, 20000 and 40000 Genome equivalents. The samples are treated according to example 10. Approach A shows one Product of 142 bases in length, approach B the same product and little product with one Length of 271 bases, approach C shows the same product quantities and approaches D and E only show the product with 271 bases in length. The amount of genome equivalents from the Sequence pBRT7QβSK145-102-431 is approximately 4000 or the equivalent of 16000 per ml.  

Example 12 Determination of a Viral Sequence Using a 5'-Nuclease Assay (Taqman Assay)

Starting from the plasmid pBRT7QβSKHCV (sequence 20) according to Example 7 Ribonucleic acid obtained from 100 ul phage supernatant. After reverse transcription a sample in the PCR with 50 pmol each of the oligonucleotide primers HC3 and HC4 and additionally with 10 pmol of the double fluorescence-labeled probe HCTM, 5'-6FAM-CAA ATC TCC Agg CAT TgA gCg ph (sequence 25, 6FAM- = 6-fluorescein-O-5'-; XT = dT-5- (CH2) 6-NH- Tetramethylrhodamine; ph = 3'-O-phosphate) added (35 cycles 1 min 94 ° C, 1 min 59 ° C, 1 min 72 ° C, 1 u AmpiTaq Gold (Perkin Elmer), ROX buffer according to the manufacturer, 100 µM dNTPs, PE7700 thermal cycler, Perkin Elmer). During the amplification, the probe HCTM partially hydrolyzed and the fluorescence of the Fluorescein increases depending on the amount of template used from the 27th to 33rd PCR cycle clus by leaps and bounds. This increase in fluorescence indicates the emergence of a PCR product after.

Example 13 Preparation of a plasmid with a sequence section from a cytokine gene

With the polymerase chain reaction with the oligonucleotide primers FAS5, 5'-gCgC AgcTT TTC TgC CAT AAg CCC TgT CC AgC ATg CCA CgT AAg CgA AA (sequence 26) and FAS3, 5'-ACCg TCTAGA Ag AAg ACA AAg CCA CCC CAA TgAgATCCAgCTgCCAgCg (Sequence 27) on the template Lambda DNA (2 µg, Boehringer Mannheim) an about 380 Base pair large fragment generated (40 pmol primers FAS5 and FAS3; 100 µM dNTPs, 10th mM Tris-HCl, pH 8.3, 2 mM magnesium chloride, 2 u Taq DNA polymerase (Hoffmann La-Roche) amplified in a volume of 50 ul. The reaction conditions are 30 cycles with 1 min at 95 ° C, 1 min at 63 ° C and 2 min at 72 ° C and finally 20 min at 72 ° C (Perkin Elmer GeneAmp 2400). The fragment is labeled with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer) digested, extracted with phenol and precipitated with ethanol.  

The plasmid pBRT7Qβd with the restriction enzymes Xba I and Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and extracted with ethanol precipitated.

The cut plasmid and the cut amplification product are combined ligated, E coli are transformed. Plasmid DNA is prepared and by DNA Sequencing verified. The plasmid pBRT7QβFas contains the Q-beta phage genome an insertion of 369 base pairs in the Hind III interface sequence 28). Phage particles are produced according to Example 6.

Example 14 Quantification of a cytokine messenger RNA

This is a protein from the tumor necrosis factor (TNF) family. The protein is on the Apoptosis involved. The amount of mRNA for fas can be interesting for medical reasons be. The sequence of the mRNA for fas is stored in the gene bank under the number M67454.

Human cells from the cell culture are mixed with different amounts of protein-coated RNA offset from the vector pBRT7QβFas. They contain an RNA that is used for the RT-PCR with the mRNA can compete for fas. The cells are disrupted and the RNA is isolated (RNAeasy, QIAGEN, according to the manufacturer). The RNA is reverse transcribed (M-MuLV, peqlab, according to the manufacturer) and amplified by PCR. The Oligonucleotide primers used are 82689, 5'-TTC TgC CAT AAg CCC TgT CC (sequence 29) and 82690, 5'-AgA AgA CAA AgC CAC CCC AA (sequence 30). The PCR product from the natural mRNA has a length of 368 base pairs, the product of the competitive RNA from the phage is 369 base pairs in length. The products let themselves through restriction digestion with the enzyme Sph I or by differential hybridization differentiate. With equal amounts of amplification product from the natural sequence and of the competitor sequence there are equal amounts of RNA in the cells and in the competitive sample.

Example 15 Preparation of a plasmid with an aptamer sequence

The plasmid pBRT7Qβd is mixed with the restriction enzyme Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and precipitated with ethanol. The oligonucleotides APT5, 5'-phosphate-AGCTTGGAGC TCAGCCTTCA CTGCATGATA AACCGATGCT GGGCGATTCT CCTGAAGTAG GGGAAGAGTT GTCATGTATG GGGGCACCAC GGTCGGATCC TA (sequence 31) and APT3, 5'-phosphate-AGCTTAGGAT CCGACCGTGG TGCCCCCATA CATGACAACT CTTCCCCTAC TTCAGGAGAA TCGCCCAGCA TCGGTTTATC ATGCAGTGAA GGCTGAGCTC CA (sequence 32) hybridized and ligated to the cut plasmid pBRT7Qβd. E. coli are transformed and plasmid DNA is prepared and verified by DNA sequencing. The plamid Product pBRT7QβAPT contains the sequence 5'-GGAGC TCAGCCTTCA CTGCATGATA AACCGATGCT GGGCGATTCT CCTGAAGTAG GGGAAGAGTT GTCÄTGTATG GGGGCACCAC GGTCGGATCC T in its Hind III interface. Become phage particles prepared according to Example 6. The RNA with the same sequence 5'-GGAGC UCAGCCUUCA CUGCAUGAUA AACCGAUGCU GGGCGAUUCU CCUGAAGUAG GGGAAGAGUU GUCAUGUAUG GGGGCACCAC GGUCGGAUCC U (sequence 33) binds specifically on L-arginine [Geiger at al., Nucleic Acids. Res., (1996) 6, 1029-1036].

Example 16 Generation of a plasmid with a degenerate aptamer sequence

The plasmid pBRT7Qβd is mixed with the restriction enzyme Hind III (Boehringer Mannheim, according to the manufacturer) cut with phenol and precipitated with ethanol.

The oligonucleotides APT5, 5'-phosphate-AGCTTGGAGC TCAGCCTTCA CTGCATGATA AACCGATGCT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ATGTATG GGGGCACCAC GGTCGGATCC TA (sequence 34) and APT3, 5'-AGCTTAGGAT CCGACCGTGG TGCCCCCATA CATNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNAGCA TCGGTTTATC ATGCAGTGAA GGCTGAGCTC CA (Sequence 35) are hybridized and ligated to the cut plasmid pBRT7Qβd. E. coli transformed and plasmid DNA is prepared and verified by DNA sequencing. The product pBRT7QβAPT contains the sequence 5'-GGAGC TCAGCCTTCA CTGCATGATA AACCGATGCT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ATGTATG GGGGCACCAC GGTCGGATCC T in their Hind III interface. Become phage particles prepared according to Example 6. The RNA is extracted from the Phages isolated. From the RNA, which contains degenerate areas, those with special Binding properties are isolated.  

Example 16 Binding of aptamers to a hapten and detection of an aptamer

Phage particles obtained with the plasmid pBRT7QβAPT are extracted with phenol and with Ethmol. The RNA contains an aptamer sequence that contains the amino acid arginine binds. D-arginine agarose or L-arginine agarose (0.1 ml) is mixed with a solution containing 0.5 µg RNA in 1 ml buffer (250 mM NaCl, 50 mM Tris-HCl, pH 7.6, 5 mM magnesium chloride) added, incubated for 10 min and washed with 300 ml of buffer. The agarose is mixed with 1 ml of buffer eluted at 95 ° C. The eluate is analyzed for the presence of the RNA by means of RT-PCR (M-MuLV, peqlab, according to the manufacturer). For this, the oligonucleotide primers 82808, 5'-gAT CCA gAA CCC gAC CgC (sequence 36) and 82809, 5'-CCA TCg gCg TgA TAg gCC (Sequence 37) used. It only arises when starting with the eluate from the L-Argi nin agarose is a 764 bp DNA fragment that demonstrates the binding of RNA to L-arginine.

The examples mentioned serve only to further explain the present invention and do not limit them in any way.  

SEQUENCELISTING

Claims (13)

1. Protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA, characterized in that the natural nucleic acid sequence is varied by singular or multiple insertion and / or substitution, these inserted or substituted sequences comprising 20 to 900 bases. 2. Protein-coated polyribonucleic acids according to claim (1), characterized in that the inserted sequences or those resulting from substitution from synthetic sequences, genomic sequences from other organisms or the variation of such sequences consist. 3. Protein-coated polyribonucleic acids according to claims (1) and (2), thereby characterized in that the genomic sequences are sections from the genome of RNA viruses, in particular sections from the RNA genome of HIV, HCV, HGV, Influenza, Picorna, Flavi, Yellow Fever, Hanta or Dengue viruses. 4. Protein-coated polyribonucleic acids according to claim (1) to (3), characterized records that it is the phage Q-beta, in its nucleic acid sequence the rear Section of the gene for the "readthrough" protein a1 in whole or in one or more Is substituted by sequences of approximately the same length in each case. 5. A method for producing a protein-coated polyribonucleic acid according to claim (1) to (4), characterized in that

• a) in a RNA virus or RNA phage genome, which is cloned as a DNA sequence, by singular or multiple insertion or substitution inserts a freely definable nucleic acid sequence of a length of 20 to 900 bases and
• b) the known genome sequence thus obtained is introduced by known methods into a suitable host cell in which viruses or phages are produced which represent the desired protein-coated polyribonucleic acid.

6. The method according to claim (5), characterized in that the host cells for the Phage Q-beta carrying a variation in the gene for readthrough protein a1 by Gene copy on a plasmid will be capable of functional failure of this To complement gene product in the varied phage. 7. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA whose natural nucleic acid sequence is varied as the standard for diagnostic procedures in which the presence of a specific ribonucleic acid is proven. 8. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA, the natural nucleic acid sequence of which is varied, as standard or Competitor sequence for processes in which the amount of a defined ribonucleic acid is determined.   9. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA, the natural nucleic acid sequence of which is varied, positive Control for the detection of viral RNA, characterized in that the protein-coated Polyribonucleic acid added directly to the sample to be determined and parallel to the Vi rus RNA is isolated. 10. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA whose natural nucleic acid sequence is varied for Control the efficiency of processes for the purification of ribonucleic acids or for Control of the efficiency of reverse transcription of ribonucleic acids. 11. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA whose natural nucleic acid sequence is varied as Comparative substance in test procedures in which nucleic acids by hybridization detected or in test procedures in which nucleic acids after or during a Amplification of the nucleic acid can be detected. 12. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA whose natural nucleic acid sequence is varied as Carrier substance for RNA sequences that have a functional property. 13. Use of protein-coated polyribonucleic acids containing a virus RNA or a bacteriophage RNA, the natural nucleic acid sequence of which is varied, as a carrier for mixtures of RNA sequences from which individual RNA sequences are selected can.