Patent Application
20080075662
The method described hereinafter makes it possible to determine, by specific hybridization and in a single step, the percentage of each of the thirty-two forms of the 5-HT2c-R mRNA even if amplification of the information contained in these mRNAs was. necessary.
After mRNA editing, the As are replaced with Is which, in turn, are replaced with dGs in the cDNA. In other words, the greater the number of As converted to Is during the initial mRNA editing, the higher the temperature required for specific hybridization. The replacement of the dGs with dis makes it possible to perform the hybridizations at a single temperature according to the strategy described below.
If necessary, if the amount of starting mRNA is too small, the information amplified in vitro can be done so in the form of single-stranded RNA, in a linear manner by in vitro transcription (IVT); it can also be done so in the form of double-stranded DNA by RT-PCR, after reverse transcription (RT) of the mRNA followed by exponential amplification by PCR. Whether the information is amplified in the final form of a single-stranded RNA or in that of a double-stranded DNA, there is the problem of identifying the Is since, in all cases, during the synthesis of the cDNA by means of reverse transcriptase, a dC is incorporated opposite each I and, during the synthesis of the complementary strand, a dG is then incorporated opposite each dC. This amounts to saying that if the sequences which are hybridized to the various probes are those which are complementary to the initial mRNAs, they contain dTs or Us opposite each unedited A, and dCs or Cs opposite each 1, according to whether the sequences were obtained in the form of DNA by RT-PCR or in the form of RNA by IVT.
When the information contained in the starting mRNAs is amplified by IVT of the sense strand, the RNA molecules synthesized have a sequence identical to that of the mRNAs, but in which the As, edited to Is, are replaced with Gs. Conversely, the IVT of the antisense strand makes it possible to synthesize RNA molecules in which the sequence is complementary to that of the starting mRNAs, but with Cs, which replace the Us opposite the sites edited from A to I.
Consequently, in order for the hybridization temperatures to be identical for all the probes, the single-stranded DNAs deposited in the form of probes on the support have a sequence identical to those of the initial mRNAs, but in which dAs replace the unedited As, and dis replace Is originating from the editing of these As to Is. Thus, whether the initial RNAs were amplified by RT-PCR in the form of double-stranded DNA or by lVr in the form of antisense RNA from the initial mRNAs, there will always be the formation of two hydrogen bonds between dA and dT or U, on the one hand, and between dl and dC or C, on the other hand.
The sequences of the thirty-two probes required for identifying the various edited or unedited forms of the 5-HT2c-R mRNA are reported below.
The Tm values having been measured individually for each of the probes, all the conditions other than the temperature being identical moreover, the hybridization temperature for all the probes is chosen between the two extreme Tm values measured; in this case, for the probe of sequence SEQ ID No. 1 and the probe of sequence SEQ ID No. 32.
The conditions other than the Tm will have been defined such that the difference between the two extreme Tm values measured is as small as possible.
The various initial forms of the 5-HT2c-R mRNA are quantified by measuring either the radioactivity or the fluorescence intensity of the thirty-two sequences paired. Since the sum of the thirty-two values measured is directly proportional to the sum of the various initial forms of the 5-HT2c-R mRNA, the percentage of each of the forms of this mRNA can be deduced by dividing the value measured individually for each form by the sum of the values of the thirty-two forms.
The Tm values having been measured individually for each of the probes, all the conditions other than the temperature being identical moreover, the inventors thus demonstrated that it was possible to very significantly reduce the difference obtained between the two extreme Tm values measured (ΔTmext) for the probe of sequence SEQ ID No. 1 and the probe of sequence SEQ ID No. 32, compared with the difference obtained between the two extreme Tm values measured for the normal probes (array of probes of the invention in which the dis are replaced with dGs).
for the array of probes of sequences SEQ ID Nos. 1 to 32 of the invention:
for the array of normal probes (without dls):
The inventors also demonstrated that, at the hybridization temperature which will be chosen for all the probes of the invention (between 61.1 and 54.13° C., preferably in the region of the mean value of 57.6° C.), the probe SEQ ID No. 32 of the invention does not hybridize with an oligonucleotide complementary to this probe, with the exception of the base located in the position complementary to the dl located at position 15 of the probe SEQ ID No. 32 (Tm of 49.4° C.).
The example of implementation developed below concerns the mRNA editing with, for application, editing of the mRNAs of one of the serotonin receptors. This example shows that it is possible to perform a hybridization under single temperature conditions, even when the percentage of guanines and cytosines initially paired goes from less than 30% to close to 60%, in a short sequence.
Five adenosines can be potentially edited in three different codons, which gives 25=32 different combinations of mRNA sequences and 3×4×2=24 different combinations of amino acid sequences (see scheme below). The deamination of the adenosines at the five sites that can be edited, given the notations A to E (or alternatively A, B, C, D and C′), results in the replacement of three amino acids of the second intracellular loop of 5-HT2C-R. The 5-HT2C-R synthesized by translation of the unedited mRNA (SEQ ID No. 34) consists of the amino acids I-N-I (isoleucine-asparagine-isoleucine) in the second intracellular loop, whereas that synthesized by translation of the completely edited mRNA (SEQ ID No. 35) or mRNA edited simultaneously at the A, E, C and D sites consists of the amino acids V-G-V (valine-glycine-valine), the codons IUI or IUA both specifying the incorporation of a valine, if I is effectively read as G. Since the codons AUI, AIU and IAU specify respectively the incorporation of M (methionine), S (serine) and D (aspartic acid), twenty-four combinations of amino acids are effectively possible in the second intracellular loop of 5-HT2c-R, for thirty-two combinations of different sequences of its mRNA.
The second intracellular loop of 5-HT2c-R is involved in the coupling of the receptor to trimeric Gq proteins. A 5-HT2c-R (I-N-I) synthesized by translation of the unedited mRNA exhibits constitutive coupling activity and therefore permanent activation of phospholipase Cβ, whereas a 5-HT2c-R (V-G-V) synthesized by translation of a completely edited mRNA responds less well to stimulation by serotonin due to a lack of coupling to trimeric Gq proteins. In other words, according to its degree of editing, 5-HT2c-R will exhibit a greater or lesser ability to respond to stimulation by serotonin.
Being able to measure the percentage represented by each of the thirty-two possible forms of mRNA amounts to predicting the percentage of each of the twenty-four forms of 5-HT2c-R and, consequently, to being able to indirectly estimate the effectiveness of 5-HT2c-R response to stimulation by serotonin. Two methods were used to estimate the percentages of the various forms of 5-HT2c-R mRNA. These two methods do not give equivalent information.
The first method, which is technically the most laborious and the most expensive, makes it possible to statistically estimate the percentage of each of the thirty-two forms of the 5-HT2c-R mRNA. It consists in simultaneously synthesizing all the cDNAs of the 5-HT2c-R mRNAs by means of specific primers common to the various mRNA forms, and then in inserting all these cDNAs into plasmids which are then transfected into bacteria. After transformation and selection, these bacteria are then cloned. These cloned bacteria are then amplified and the plasmid DNA is then extracted therefrom and purified and the inserted cDNAs are then sequenced. If the number of cDNAs cloned and sequenced is sufficient to be statistically representative of the initial population of cDNA, and therefore of mRNA, the percentage of each of the initial forms of 5-HT2c-R mRNA may then be deduced. If this number is insufficient, only the major forms of the 5-HT2c-R mRNA will be statistically represented.
The second method can be carried out more rapidly but it does not make it possible to estimate the percentage of each of the thirty-two forms of the 5-HT2c-R mRNA. On the other hand, it makes it possible to determine the percentage of each A edited to 1, at each of the five sites that can be edited. For this, after synthesis of the cDNAs as in the preceding case, a primer extension is carried out in the presence of three of the four usual deoxyribonucleoside triphosphates and of a dideoxyribonucleoside triphosphate which terminates the primer extension opposite the deoxyribonucleoside whose nature it is desired to determine. For example, the three deoxyribonucleoside triphosphates may be dATP, dGTP and dTTP, while the dideoxyribonucleoside triphosphate is ddCTP (dideoxycytidine triphosphate) which, by incorporating itself opposite the first dG encountered, stops the primer extension at the position where there was an I in the mRNA which served as a matrix for the cDNA synthesis. When the initial adenosine has not been edited and therefore replaced with a dG in the cDNA, the primer is extended until the first dG which follows the dA originating from the unedited adenosine. Under these conditions, the primers must be judiciously chosen, complementary to one or other of the two strands of the cDNA, and must also have been labeled with 32P in the 5′ position, in order to be visualized by autoradiography after separation by polyacrylamide gel electrophoresis. Measurement of the radioactivity contained in the various separated fragments makes it possible to estimate the percentage of As edited at each of the five sites that can be edited. At least as many primers as there are sites that can be edited are necessary.
To extract the RNAs from frozen tissues in the RNA Later solution (appendix C), or freshly dissected tissues, these tissues are ground in 350 μl of the RA1 solution of the NucleoSpin RNA II extraction kit (appendix B1), to which 3.5 μl of β-mercaptoethanol have been added (appendix C). The lysate is filtered by centrifugation on a filtration column (appendix B1) at 11000×g for 1 minute. After homogenization of the filtrate with 350 μl of a 700 ethanol solution, the entire mixture is centrifuged on a Nucleospin RNAII silica column (appendix B1) for 30 seconds at 8000×g. The column is then washed using 350 μl of the MDB (membrane desalting buffer) solution (appendix B1) and centrifugation for 30 seconds at 11000×g. In order to remove any possible contaminating genomic DNA, 10 μl of the DNase I solution (appendix B1) are placed on the column for 30 minutes at ambient temperature. After complete digestion of the DNA, the DNase I is removed by washing with 200 μl of the RA2 solution (appendix B1) and then further washing with 600 μl of the RA3 solution (appendix B1), each wash being followed by centrifugation for 30 seconds at 8000 g. The RNA attached to the column is washed a final time with 250 μl of the RA3 solution (appendix B1) and finally dried by centrifugation for 2 minutes at 11000 g. The RNA is eluted with 60 μl of RNAse-free water (appendix B1), after centrifugation for 1 minute at 11000 g.
For this step, the ThermoScript RT-PCR system kit and protocol (appendix B2) are used. For each RNA sample, 500 ng of total RNA are mixed with 1 μl of poly-(dT)20 (50 μM; appendix B2), optionally made up to a final volume of 10 μl with RNase-free water (appendix B2). This volume is incubated for 5 minutes at 65° C. and is then immediately cooled in ice. 4 μl of the cDNA synthesis buffer (5×concentrated; appendix B2), 1 μl of the DTT solution (DTT for dithiothreitol) (0.1 M; appendix B2), 1 μl of RNase OUT (40 U/gl; appendix B2), 1 μl of RNase-free water (appendix B2), 2 μl of a mixture of dNTP (dNTP: deoxyribonucleotide triphosphate) (10 mM each; appendix B2) and 1 μl of the thermoScript RT enzyme (15 U/μl; appendix B2) are then added, and the entire mixture is incubated at 50° C. for 60 minutes. The reaction is then stopped by heating the reaction mixture at 85° C. for 5 minutes. To remove the RNA, the reaction medium is incubated in the presence of 1 μl of RNase H (appendix B2) at 37° C. for 20 minutes.
The example here is given specifically for the 5-HT2C-R gene. Those skilled in the art may readily transpose or adapt such a PCR amplification protocol to other mRNAs which may be edited by choosing the pair of primers for specifically amplifying the mRNA fragment comprising the editing sites.
The pair of primers PCR9, PCR10, used for the PCR amplification of a fragment of the 5-HT2c-R gene, was chosen in DNA regions that are identical in humans, mice and rats. The sequences of the pair of primers are:
The resulting PCR amplification product is 250 base pairs in size, in the 3 species human, mouse and rat.
For the demonstration of SSCP (single strand conformation polymorphism), these primers can be labeled with various fluorophores such as C6-FAM or HEX.
For the PCR amplification step (PCR for “Polymerase Chain Reaction”), the Platinum Taq DNA polymerase protocol and kit (appendix B3) are used. For the PCR amplification of a region that includes the 5-HT2c-R gene editing site, the reaction mixture comprises 2 μl of the cDNA solution, 5 μl of the amplification buffer (10 times concentrated; appendix B3), 1.5 μl of an MgCl2 solution (50 mM; appendix B3), 1 μl of a mixture of the four dNTPs (10 mM each; appendix B3), 1 μl of each of the primers PCR9 and PCR10 (solution at 10 μM), 0.5 μl of the Platinum Taq enzyme (5 units per ml; appendix B3), made up to 50 μl with sterile distilled water. This reaction volume is initially heated in a PTC200 thermocycler (appendix A) for 2 minutes at 94° C. It is then subjected to 30 cycles comprising phases of denaturation at 94° C. for 30 seconds, and then of hybridization for 30 seconds at 65° C. decreased by 0.3° C. for each subsequent cycle, and of elongation at 72° C. for 30 seconds. A final elongation step is carried out at 72° C. for 6 minutes.
For this purification step, the Nucleospin Extract protocol and kit (appendix B4) are used. The reaction volume obtained by PCR (50 μl) is mixed with 200 μl of the NT2 solution (appendix B4) and directly loaded onto the purification column. After centrifugation at 11000 g for 1 minute, the PCR amplification products attached to the column are washed using 600 μl of the NT3 solution (appendix B4) and centrifugation at 11000 g for 1 minute. A final wash is carried out with 200 μl of the NT3 solution (appendix B4) and the column is then dried by centrifugation at 11000 g for 2 minutes. Elution of the DNA is obtained by incubating the column with 50 μl of the NE elution buffer (appendix B4) at ambient temperature for 1 minute, followed by centrifugation at 11000 g for 1 minute.
The DNA 1000 Assay kit and protocol (appendix B5) and the 2100 bioanalyzer (appendix A) are used to determine the concentration of DNA obtained after PCR amplification and column purification. The gel is prepared by vigorously mixing an aliquot of gel (appendix B5) with 25 μl of dye (appendix B5). This matrix is then centrifuged at 2400 g for 15 minutes and is then injected (9 μl) into the microcapillaries of the DNA chip (appendix B5). For the quantification, the PCR DNA samples (1 μl) are loaded into the corresponding wells, in the presence of an internal size indicator (5 μl; appendix B5) and of another size marker (1 μl; appendix B5). The electrophoresis and the calculation of the concentration of DNA are carried out by running the BioSizing program of the 2100 Bioanalyzer (appendix A).
The analysis of the PCR-amplified DNA labeled on each of the 2 strands with fluorophores of C6-FAM and HEX type (appendix C) is carried out by capillary electrophoresis on the ABI PRISM 3100 device (appendix A). The DNA samples to be analyzed are first of all diluted in water to a concentration of 250 pg/μl. For each sample, a mixture is prepared that contains 1 μl of the purified and diluted PCR product solution, 0.5 μl of GeneScan 500 ROX migration standard, 0.5 μl of an NaOH solution (0.3 N) and 10.5 μl of formamide. This mixture is brought to 90° C. for 2 minutes and is then immediately cooled in ice. The capillary electrophoresis begins with a premigration of 3 minutes at 15000 volts. The DNA is injected at 1000 volts, for a period of 22 seconds. Finally, the electrophoresis of the DNA is carried out at 15000 volts and 18° C. in a one-times concentrated TBE buffer (TBE for Tris-Borate-EDTA) containing the GeneScanPolymer gel (5%) and glycerol (10%). At this stage, the fluorescence reading and the data acquisition are carried out automatically. These data are visualized and processed using the GeneScan analysis program.
The results make it possible in particular to obtain an editing profile of said mRNA under given conditions (length of the PCR products, conditions of the above steps of the SSCP protocol, parameters for the data processing, etc). The parameters of this profile (such as the migration time, the height and the surface area of the peaks observed on this profile) will be or may be compared to standard profiles, for instance:
to the characteristic profile obtained under these given conditions for each of the separate edited (or unedited) forms of said mRNA (cf. for example
to profiles corresponding to known qualitative and/or quantitative mixtures of each of these edited or unedited forms, obtained under given conditions; and/or
to known editing profiles of the same mRNA for normal patients or patients presenting confirmed pathologies, for mRNA extracts of specific tissues (cf. for example
For each of these “standard” profiles stored in the memory of the analysis program and obtained under these given conditions, the percentage and/or the rate of editing for each of the forms may also be known.
To obtain the characteristic profile of each of the edited (or unedited) forms of said mRNA by SSCP under given conditions (steps 1 to 6 in Example 3), the following process may be carried out:
cloning and verification by sequencing of the edited or unedited form for which it is desired to obtain the characteristic profile;
PCR amplification of the identified and cloned sequence (step 3 of Example 3);
implementation of steps 4 to 6 of Example 3.
In order to determine the editing percentages and/or the editing rate for standard profiles corresponding to known mixtures of edited or unedited forms, it is sufficient to mix, before step 3 of the above process, in the desired proportions, the editing forms included in the mixture.
To determine the editing percentages and/or rate for standard profiles corresponding to reference editing profiles which may be in the memory of the analysis system, it being possible for these profiles in particular to correspond to editing profiles obtained for normal patients or patients exhibiting a confirmed pathology, for a particular tissue, before or after therapeutic treatment, etc., it will be sufficient to clone and sequence a representative portion of or all the sequences of said mRNA present in the mRNA extract and to deduce therefrom the proportion of each of the forms present.
In another method, it will also be possible to use the biochips of the invention on which all the editing forms of said mRNA will be deposited.
ABI Prism 3100 (Applied Biosystems);
2100 Bioanalyzer (Agilent Technologies; No. DE13701290);
Eppendorf Centrifuge 5415 D (serial No. 5425 39 178); and
MJ Research PTC200 Thermocycler (serial No. AL046013 and EN015975).
1—NucleoSpin RNA II (Invitrogen; reference: 740.955.50);
2—ThermoScript RT-PCR system (Invitrogen; reference: 11146-024);
3—Platinum Taq DNA polymerase (Invitrogen; reference: 10966-026);
4—Nucleospin Extract (Macherey-Nagel; reference: 740.588.50); and
5—DNA 1000 Assay (Agilent Technologies; reference: 5065-4449).
β-mercaptoethanol (Sigma; reference: M 3148);
primer PCR 9 (Proligo; 5′-labeled with the fluorophore C6-FAM and purified);
primer PCR 10 (Proligo; 5′-labeled with the fluorophore HEX and purified); and
10×TBE (Invitrogen; reference: 15581-044).
1) Bringing said compound to be evaluated into contact with a population of eukaryotic cells, in particular murine cells, expressing the gene of said mRNA (in vitro, in cellulo or in vivo)
Intraperitoneal injection of Balb C mice with the compound to be tested, approximately 20 mg/Kg.
At given times, for example up to 3 days: the mice are killed and a sample of specific tissue, such as the prefrontal cortex, is removed, frozen and then microdissected.
2) Demonstration of the modulation of the editing of said mRNA fragment in the cells using an mRNA extract, by means of the pair of primers PCR9 (SEQ ID No. 36) and PCR10 (SEQ ID No. 37).
cf. Example 3.
3) Comparison of the editing rate and/or of the editing profile obtained with that obtained for an mRNA extract derived from the same population of cells from control mice that have not been brought into contact with the compound to be evaluated, and demonstration of the modulation or non-modulation of the editing rate and/or profile.
4) Comparison of the editing rate and/or of the editing profile obtained with the standard profiles obtained under the same analytical conditions for identical cell populations after treatment with a compound known to modulate the editing rate and/or profile of said mRNA, and the therapeutic effect of which is known, and selection of said compound tested as a potential agent for exerting the same therapeutic effect if the modulation observed for the product tested is similar to that observed for the known compound.
It was possible to demonstrate that a compound known to be an active antidepressant was capable of modulating the 5-HT2c-R editing rate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02/09,524 | Jul 2002 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR03/02339 | 7/24/2003 | WO | 00 | 1/25/2005 |
