EP0702690A1

EP0702690A1 - Freeze-dried compositions comprising rna

Info

Publication number: EP0702690A1
Application number: EP95915190A
Authority: EP
Inventors: Marcelus Hendrikus Franciscus Uylen; Franciscus Henricus Maria Van Dinther; Tim Kievits
Original assignee: Akzo Nobel NV
Current assignee: Akzo Nobel NV
Priority date: 1994-04-07
Filing date: 1995-04-07
Publication date: 1996-03-27
Also published as: WO1995027721A1; JPH08511956A; AU2215995A

Abstract

The invention relates to freeze-dried particles comprising ribonucleic acid (RNA) and methods for the preparation of said particles. The invention further relates to the use of said particles in methods for the detection of nucleic acid as well as test-kits for the detection of nucleic acid comprising said particles. By freeze-drying RNA, a stable product can be obtained that can be preserved for a long time without loosing its structure. The freeze-dried RNA-containing particles according to the invention are therefore very useful alternatives for, for example, presently used RNA-containing reagent solutions. The particles according to the present invention are dry and therefore almost insensitive to various degradation processes.

Description

FREEZE-DRIED COMPOSITIONS COMPRISING RNA

The invention relates to freeze-dried compositions comprising ribonucleic acid (RNA) and methods for the preparation of said compositions. The invention further relates to the use of said compositions in methods for the detection of nucleic acid as well as test-kits for the detection of nucleic acid comprising said compositions.

RNA is known to be a rather sensitive and relatively unstable entity. RNA in solution is subject to more or less rapid deterioration. Especially degradation by RNase is a major concern when handling RNA preparations. This omnipresent enzyme (RNase is present on fingertips, in dust etc.) is heat stable and does not require cofactors for its enzymatic activity. This makes it difficult to inactivate RNase in RNA preparations. Although there are several isolation methods yielding RNase-free RNA (Sambrook J., Fritch E.F. and Maniatis T. (1989) Moecular Cloning: A Laboratory Manual, 2nd edn Vol.1 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor; Ausubel, F.M. et al. (eds) (1990) Current Protocols in Molecular Biology, Vol.1 , Green/Wiley-interscience, New York), precautions have to be undertaken to avoid RNase contamination at the final step of a purification or isolation, i.e. solubilization of RNA. The common practice is to solubiiize RNA in diethylpyrocarbonate (DEPC)-treated water (Chomczynski P.(1992), Solubilization in formamide protects RNA form degradation. Nucleic Acid Research, Vol.20(14)). The solubilized RNA is susceptible, however, to accidental contamination during handling and storage of samples. In addition, long-term storage of water solubilized RNA samples requires temperatures as low as -70 °C to prevent degradation. As an alternative, RNA can also be solubilized in formamide instead of DEPC-treated water. Formamide effectively protects RNA from degradation by RNase, and allows for long-term storage at -20°C.

However, for an RNA preparation to be turned into a viable commercial product, with reasonable shelf-life capable of being shipped and stored at 2-8°C or even higher temperatures, above described methods do not suffice. The need therefore exists for a stable RNA-containing composition which can easily be preserved for a long time and is easy to handle.

The present invention provides freeze dried RNA-containing particles.

It has been found that the freeze-drying process has no negative effect on the RNA and substantially retards all possible inactivation processes.

CONFIRMATION COW By freeze-drying RNA, a stable product can be obtained that can be preserved for a long time without loosing its structure. The freeze-dried RNA-containing compositions according to the invention are therefore very useful alternatives for, for example, presently used RNA-containing reagent solutions. The particles according to the present invention are dry and therefore almost insensitive to various degradation processes.

The present invention can be used with a wide diversity of RNA's. RNA sequences of different lenghts can be freeze-dried. All RNA sequences varying in length from, for example, a few hundred up to thousands of nucleotides can be incorporated in the freeze-dried compositions according to the present invention.

The freeze-dried particles according to the present invention can be presented in different paniculate forms.

Surprisingly, said particles have a stability that is greater then material that is freeze dired in, for example, layers, and are highly soluble. RNA in these freeze-dried particles is stable during storage for at least one year at 2-8°C up to 37°C. Preferably the RNA is freeze-dried in the form of a spherical particle. Spherical freeze-dried particles (accuspheres or lyospheres) are known from Price et al. (US 3,655,838). These spherical beads, contain material for immunological reactions. Lyospheres comprising biological active materials are known, for instance from USP 3,932,943, and also from many other patents. Advantages of lyospheres are the uniformity of the particles, the easy to handle products, the faster freeze- dry process, less degradation during the freeze-dry process, and the improved dissolution properties.

The lyospheres can be prepared by freeze-drying droplets of an aqueous RNA- solution. To obtain droplets of the required size, the solution can be sprayed into a cold bath (for example as disclosed in DT 2,140,747, EP 8191 3, or US 3,928,566), into liquid nitrogen (for example as disclosed in J5 91 69,504), or onto a refrigerated drum (for example as disclosed in USP 3892876) or a refrigerated plate (for example as disclosed in US 4,501 ,719). Various other methods are well- known in the art.

The freeze dried particles according to the present invention may comprise, in addition to the RNA, stabilizers such as polyhydroxy-compounds like sucrose, mannitol, dextran, sorbitol and glycerol. The freeze-dried particles according to the present invention are especially useful as diagnostic reagents in methods for the detection of nucleic acid. Diagnostic methods for the detection of nucleic acid usually involve the amplification of nucleic acid, isolated from a clinical sample.

One of the amplification techniques which can be used with the freeze-dried particles according to the present invention is NASBA, the "nucleic acid sequence based amplification (NASBA)" as disclosed in European Patent application EP 0,329,822. With this technique large amounts of RNA can be generated.

In diagnostic methods for the detection of nucleic acid sequences, comprising the amplification of a target sequence, an internal control sequence is sometimes added to the sample. Such a method has been described in co-pending, co-owned application no. PCT/EP93/02248. An internal control sequence as used with the method described in PCT/EP93/02248 comprises a nucleic acid sequence distinguishable from the target sequence that can be amplified with the same amplification reagents as the target sequence.

These "internal control sequences" can be incorporated in freeze-dried particles according to the present invention, which are then suitable for use with a method as described in PCT/EP93/02248.

Likewise with methods for the quantitative determination of nucleic acid in a sample the freeze-dried particles can be used.

A method for the quantification of nucleic acid, using the addition of a known number of molecules of a nucleic acid sequence, corresponding to the target nucleic acid, to a sample containing an unknown amount of the target nucleic acid sequence has been described in co-pending co-owned European patent application published under no. EP 525882. The method as described in EP 525882 is based on the principle of amplification of nucleic acid from a sample containing an unknown concentration of wild-type target nucleic acid, to which has been added a known amount of a well-defined mutant sequence. Amplification is performed with one primer set capable of hybridizing to the target as well as the mutant sequence. The competitive amplification described in EP 525882 can be performed with a fixed amount of sample and dilution series of mutant sequence or vice versa. The mutant sequence can again be incorporated in a freeze-dried particle, which might also contain the primers and probes needed for carrying out the quantitative method as described above.

A method for the quantification of analyte nucleic acid in a sample comprising the addition to a sample of different respective amounts of different nucleic acid constructs, each construct being distinguishable from the analyte nucleic acid and capable of being co-amplified with the analyte nucleic acid has been described in co-pending co-owned application no. PCT EP 94/02295. It is evident that all the RNA sequences used can be incorporated in a freeze-dried particle according to the present invention, in their respective amounts.

The freeze-dried particles according to the present invention are preferably produced in the form of spheres.

Spherical particles according to the invention are preferably produced by a method comprising the steps of preparing an RNA solution comprising one or more stabilizers and forming freez-dryed particles form said solution, preferably by dripping said solution into liquid nitrogen, keeping the droplets in the liquid nitrogen until they are completely frozen and lyophilizing said droplets.

The freeze-dried particles according to the present invention can be incorporated in a test-kit for the detection or quantification of nucleic acid, together with other reagents used with the detection method employed, such as suitable enzymes, primers and probes.

EXAMPLES:

Example 1 : Preparation of RNA-containing freeze-dried particles.

A. All chemicals used were of reagent grade 1 quality.

A stabilizer mixture was prepared using 15% w/w saccharose, 5% w/w mannitol and 5% w/w dextran T-40 in water. (The saccharose was obtained from Sigma, mannitol from Baker and Dextran T40 from Farmacia).

Separately an RNA solution was prepared by 100 times dilution in DEPC- treated water of a concentrated RNA stock. 50 ml of the stabilizer solution was mixed with 50 ml of the RNA solution.

Lyospheres were prepared from this RNA/stabilizer solution in the following way:

50 μ\ portions of the RNA/stabilizer mixture were dripped into boiling nitrogen. The spheres were kept into liquid nitrogen until they were completely frozen. The frozen spheres were lyophilized yielding spheres with a residual moisture content below 2% w/w and essentially having the same diameter as the frozen spheres. Each accusphere thus prepared contained 1.22 ^# 10⁷ RNA molecules.

B. Alternatively the RNA-containing freeze-dried particles can be prepared in the following way:

A mixture of RNA solutions was prepared for the preparation of 600 accuspheres containing 10^ 2 molecules of each species of RNA per accusphere. The solution consisted of three different RNA species each in a volume of 9ml (total volume of 27ml). The RNA solutions contained 1 ,5 M sodiumchloride, sodiumchloride was obtained from Janssen Chimica. The same stabilizers as used under A were added to the RNA mixture as dry components. 2.25 g of saccharose, 0.75 g of mannitol and 0.75 g of dextran T40 was added to a tube containing the RNA mixture. Water was added to the RNA/stabilizer mixture until a final volume of 30 ml was obtained.

Lyophilization was carried out as described under A.

Freeze-dried particles containing RNA can likewise be prepared in the following way:

A RNA solution was prepared for the preparation of 600 accuspheres containing 7.6* 10^ RNA molecules per accusphere.

3.0 gram sucrose, 1.0 gram mannitol and 1.0 gram dextran T40 were dissolved in 40.0 ml. water. 45 μ\ of the RNA stock solution containing 10^

RNA molecules per μ\ was added to 30 ml of the sucrose/mannitol/dextran

T40 containing solution.

Lyophilization was carried out as described under A.

Example 2: Stability of freeze-dried particles comprising RNA.

The stability of RNA as a freeze-dried sphere was tested by comparing the amplification efficiency of NASBA reactions using RNA from lyophilized spheres according to the invention and an independend reference. The lyophilized spheres were prepared as described under "A" in example 1 . The reference Wild-type (WT) is an in vitro generated HIV-1 cell line quantitated by means of electron microscopy as 2.9 ( ± 1 .6) x10^A10 viral particles per ml (layne et al., 1992). This reference was diluted in lysis buffer and stored as single use aliquots of 100 //I as 7300 RNA molecules per μl at -70°C.

The lyophilized spheres tested in this stability study were four different batches of RNA spheres, stored at 2-8°C, 20-25 °C and 35-38°C.

The following protocol was used to determine the stability:

An aliquot of 10 μ\ of the diluted reference was added to 900 μ\ of lysis buffer and mixed. The dried RNA sphere was diluted in 550 / I of elution buffer (1 mmol/l Tris/HCI pH 8.5), concentration of the three RNAs, Qa, Qb and Qc respectively 1000000, 100000 and 0000 RNA molecules per 50 μl of elution buffer. Qa, Qb and Qc are in vitro synthesized RNA sequences having a length of approximately 1000-1500 nucleotides. An aliquot of 50 μ\ of the dissolved RNA sphere was added to the lysis buffer tube and mixed. An isolation procedure described by Boom et al (Boom R, Sol CJA, van der Noordaa J, et al: Rapid and simple method for purification of nucleic acids, J Ciin Microbiol 1990; 28:495-503.) was used to isolate the RNA calibrators and the WT reference. Amplification was performed according to the method described by ievits et al (Kievits T, van Gemen B, Lens P, et al: NASBA^M isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection, J Vir Meth 1991 ; 35:273-286.) with minor modifications.

Detection of HIV-1 RNA in a sample is based on the electrochemi-luminescence principle (Blackburn GF, Shah HP, Kenten JH, Leland J, Kamin RA, Link J, Peterman J, Powell MJ, Shah A, Talley DB et al: Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics, Clin Chem 1991 ; 37:1534-1 539, Kenten JH, Gudibande S, Link J, Willey JJ, Curfman B, Major EO, Massey RJ: Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products, Clin Chem 1992; 38:873-879).

To separate the amplificates (WT, Qa, Qb and Qc), aliquots of the amplified sample were added to four hybridization solutions, each specific for one of the amplificates. Here, the respective amplificates were hybridized with a bead-oligo (i.e. a biotin- oligo bound to streptavidin coated magnetic beads acting as the solid phase) and a ruthenium-labeled probe. The magnetic beads carrying the hybridized amplificate/probe complex were captured on the surface of an electrode by means of a magnet. Voltage applied to this electrode triggers the electrochemiluminescence (ECL) reaction. The light emitted by the hybridized ruthenium-labeled probes is proportional to the amount of amplificate. Calculation based on the relative amounts of the four amplificates reveals the amount of WT reference.

The amount of WT reference RNA was determined according to the above listed protocol. Results are depicted in table 1.

No significant decrease of RNA, except for normal variation in the quantitative

NASBA assay, can be observed after storage of the RNA spheres up to 14 weeks at all tested temperatures (table 1 ).

These results indicate that apart from batch to batch and day to day variation there is no significant increase of the calculated WT reference amount over time and temperature which would indicate a decrease of the amount of Qa, Qb and Qc RNA in the spheres.

Table 1 :

Example 3: Stability of freeze-dried particles (2)

Accuspheres, containing in vitro synthesized RNA sequences of approximately 1500 nucleotides, prepared according to the protocol C of example 1 were dissolved in 200 //I elution buffer (10 mM Tris/HCI, pH 8.5). 20 l of this solution was added to 9ml lysis buffer (5 M guanidin thiocyanate, 1 % (v/v) Triton X-100, 20mM EDTA, 50 mM Tris/HCI, pH 6.4). Subsequently 70 μ\ activated silica (1 mg/ml size selected suspension in 0.1 N HCI) was added to the lysis mixture to bind the nucleic acid. After washing and drying the silica, nucleic acid was eluted in 100 μ\ elution buffer.

Nucleic acid amplification is performed as described by Kievits et al. with the following modifications:

The final volume of the reaction mixture is 25 μ\, containing 40 mM Tris/HCI, pH 8.5, 12mM MgCl2, 40 mM KCI, 5mM DTT, 1 mM of each dNTP, 2 mM of each NTP, 15% (v/) DMSO, 0.1 / g/μl BSA, 0.2 //I of each of the oligonucleotide primers, 8.0 u AMV-RT, 0.1 u RNase-H and 40.0 u T7 RNA Polymerase. One primer carries a T7 RNA polymerase recognition site. For each amplification reaction 8 μ\ of the isolated RNA was used (containing approximately 200 molecules of RNA). The amplification reactions are performed in centrifuge micro test tubes at 41 °C for 90 minutes. The amplifcation products are analysed by enzyme-linked gel assay (ELGA). Amplified RNA is detected by a non-radioactive hybridization in solution, using sequence specific HRP δ'-labelled oligonucleotide probes. After hybridization, non-hybridized probes were separated form the homologeous hybridized product by gel eiectrophoresis. Free HRP probes and hybridized products were directly visualized in a polyacrlyiamide gel by incubating the gel in a substrate solution for HRP.

The above described procedure was applied to three different lyophilized RNA- preparations that had been stored at 4°C, 20-25°C and 35-38 °C. Using the above described ELGA detection no differences between the signals were observed after 8 months (batch 3) and 1 year storage (batch 1 and 2). These results were confirmed using reference RNA (as described in example 2) stored at - 70°C: after co-isolation, co-amplification and detection of the reference RNA and the lyophilized RNA no significant differences were found in the ECL ratio's for the reference and lyophilized RNA, indicating that the RNA-containing accuspheres according to the invention are stable when stored at 4°C, 20-25°C and 35-38 °C. Example 4: Stability of freeze-dried particles comprising gel marker RNA.

Accuspheres, prepared according to protocol B of example 1 , containing gelmarker RNA were dissolved in 25 μl of RNase, DNase and protease free water. Added to this solution is 25 μl of a horse-radish peroxidase (HRP) 5'-labelled oligonucleotide probe solution. Hybridization of this sequence-specific oligonucleotide with the gelmarker RNA takes place a 41 °C for 15 minutes. After hybridization, non- hybridized probes were separated from the homologeous hybridized product by gel electrophoresis. Free HRP-probes and hybridized products were directly visualized in a polyacrylamide gel by incubating the gel in a substrate solution for HRP (tetramethyl benzidine, TMB).

Three different gel marker RNA accusphere lots were analyzed as described above, in a long-term stability study. The first two batches show no sign of RNA degradation upon storage of the gelmarker RNA accuspheres at 4°C for 52 weeks. The third batch was tested after 36 weeks and also showed the expected gel pattern without degradation. If degradation of RNA had occurred this would have been indicated by the appearance of RNA smears in the gel instead of the discrete bands that were found.

Claims

Claims:

1 . Freeze-dried particle comprising ribonucleic acid.

2. Freeze-dried particle according to claim 1 , additionally comprising one or more stabilizers.

3. Freeze-dried particle according to claim 2, wherein the stabilizers are saccharose, mannitol and/or dextran.

4. Freeze-dried particle according to claim 3, said particle being a spherical particle.

5. Freeze-dried particle according to any of claims 1 -4 having a moisture content of less than 2% w/w.

6. Method for the preparation of a particle according to any of claims 1 -5, comprising preparing an RNA solution comprising one or more stabilizers and forming freeze-dried particles from said solution.

7. Method for the preparation of a particle according to claim 4, comprising the steps of preparing an RNA solution comprising one or more stabilizers, dripping said solution into liquid nitrogen, keeping the droplets in the liquid nitrogen until they are completely frozen, lyophilizing said droplets.

8. Use of a freeze-dried particle according to any of claims 1 -5 in a method for the detection of nucleic acid.

9. Test-kit for the detection of nucleic acid comprising at least one freeze-dried particle according to any of claims 1 -5.