DE10324063A1 - Preparing nucleotides on controlled pore glass, useful for making libraries for screening to identify aptamers, by coupling protected deoxyphosphoramidite directly to amino groups on the glass - Google Patents

Abstract

Preparing nucleotides (I) bound to porous glass (controlled pore glass; CPG) comprising functionalizing the surface of CPG with amino groups; coupling a protected deoxyphosphoramidite (I) directly to the amino for construction of a nucleotide, then cleaving protecting groups with a solution containing an aminoalkane, optionally mixed with a base, at room temperature to 100 [deg]C for 2-20 minutes, is new. An independent claim is also included for nucleotide libraries prepared by the new method.

Description

The The invention relates to a method for producing porous glass bound nucleotides with a special method for cleaving Protecting groups and nucleotide libraries were made using this procedure.

Bruce Merrifield was the first to deal with carrier-bound syntheses in the 1960s. Long-chain-amino-alkyl-CPG (LCAA-CPG), which is loaded with the succinate of the respective base, has long been known and has been widely used for the production of oligonucleotides. This forms an ester bond to the carrier, which is used in the treatment with conc. NH _{3 is} cleaved after synthesis. This cleaves the oligonucleotide from the support. However, supports are also described which allow the oligonucleotide to remain on the support.

• 1. Die sogenannte "diskontinuierliche" Bibliothek, die eine Sequenzierung der Oligonucleotide nach Anwendung nötig macht, da die Synthese auf einem Träger stattfindet, der aus Partikeln besteht.
• 2. Die "kontinuierliche" Bibliothek, welche z.B. auf Glasplättchen oder Polypropylenstreifen hergestellt wird. Hierbei ist durch die Position des jeweiligen Oligonucleotids auf dem Träger die Sequenz festgelegt.
• 3. Die Bibliothek in Lösung.

Various types of libraries are known to modern biotechnology.

• 1. The so-called "discontinuous" library, which requires sequencing of the oligonucleotides after use, since the synthesis takes place on a support that consists of particles.
• 2. The "continuous" library, which is produced, for example, on glass plates or polypropylene strips. The sequence is determined by the position of the respective oligonucleotide on the support.
• 3. The library in solution.

At the so-called "screening" one sets oligonucleotide libraries a, in which the oligonucleotides are bound to the support after deprotection remain. An example of oligonucleotide synthesis using standard methods is with H. Köster, A. Stume, A. Wolter, Tetrahedron Lett. (1983), Vol. 24, No. 8, 747-750 described.

Likewise the synthesis of oligonucleotides on unmodified is known CPG. The disadvantage of this method is that the oligonucleotides are not more of the carrier material split off for analysis.

B. Bernard, J Jamal, F. Choplin, J. Martin, EP 1248859 report on the production of oligonucleotides on surface-modified glass, but the "spacer" used arises in a complex synthesis and contains at least one hetero atom in addition to the anchor group. This increases the cost of the process and is very time consuming since silylations have to be carried out.

U. Markos and EM Southern, Nucl. Acids Res. (1996), Vol. 24, No. 15, 3048-3052, used a complex "spacer" with oxirane ring systems. The "spacer" described here is also not as resistant to the reactive reagents used in oligonucleotide synthesis as a short CH ₂ chain, and the product, because it was produced on OH anchor groups, cannot be used for later analysis be split off in one step, as is the case with NH ₂ groups.

So-called polymer supports are still used most widely in practice, not only for "screening". The bases are coupled directly to the NH ₂ groups of the support. The most significant disadvantage of these materials are the difficulties in reproducibility with single particle selection and the poor synthesis.

B. Rotte, M. Hinz, R. Bader, A. Astriab, M. Markiewicz and WT Markiewicz and H. Seliger, Collect. Czech. Chem. Commun., (1996), 61, 304-306, described oligonucleotide syntheses according to standard methods on NH ₂ functionalized CPG, which means that the oligonucleotides cannot be deprotected without severely attacking the surface of the CPG during deprotection with ammonia and suffer a great loss of substance. The Si-O bond of the CPG is known to be base labile. If the exposure time of the ammonia is chosen too short, the reactions of the deprotection are not complete.

Of others apply polar, superficial negatively charged carriers, such as controlled pore glass (CPG) as a poor choice for "screening" polar biomolecules, such as Polynucleotides, DNA or proteins, since these, when fluorescent were marked by their strong non-specific binding to the carrier background fluorescence.

The known methods and syntheses therefore always pose the problem on either yields per coupling step of the oligonucleotide result on CPG, or, in the case of the polymer carrier, at the same time high Loading the possibility leave the oligonucleotides on solid phase for further use supply, but low coupling yields per coupling step or even incorrect couplings exhibit. With high demands on the purity of the oligonucleotide, as is the case with "screening", everyone comes across previous procedures to the limit of their performance, or deal deals with arrangements that are not suitable for "discontinuous" libraries, such as arrays, enzymatic synthesis or oligonucleotide binding to the carrier material after synthesis.

task The present invention is therefore a new method for Manufacture of porous glass indicate bound nucleotides and a corresponding nucleotide library.

The The object described above is achieved by a method such as it is defined in claim 1. The subclaims show advantageous further training.

The newly developed process involves the production of nucleotides such as polynucleotides or oligonucleotides on a carrier material, which consists of particles. This is also suitable for production of oligonucleotide libraries. Surprisingly it has been found that it is possible with the method according to the invention bind polar nucleotides to a polar support.

The The invention is described in more detail below using an example.

These are libraries of the type: Particle - TTTT - oligonucleotide library. For this purpose, the oligonucleotide is built up step by step using two differently modified supports (see 1 and 2 ).

The aminopropyl CPG used has a pore size of 1400 Angstroem. Starting from aminopropyl CPG whose ninhydrin test is positive, the triazine-modified CPG (0.2 g aminopropyl CPG, 0.27 g cyanuric chloride, 0.37 g NaHCO ₃ , 3.75 ml water, 10 ml acetonitrile) is obtained with cyanuric chloride RT 3h). The ninhydrin test of this material after washing with water and acetonitrile is negative. By coupling the triazine ring at room temperature with 1,2-diaminoethane (0.1 ml 1,2-diaminoethane, 10 ml acetonitrile at RT 3 h), a support is obtained in which the ninhydrin test responds, since it is very sensitive Represents evidence for amino groups.

The conditions of the synthesis included on both supports a modified phosphoamidite chemistry compared to the standard phosphoramidites, the corresponding phosphoamidite with acetyl protective group being used instead of the usual benzoyl-protected deoxycytidinephosphoamide ( 3 ).

Detritylation 2% TCA / 1,2-dichloroethane / 1min; Coupling 50 ul 0.1 M phosphoamidite, 200 ul 0.25 M DCI / 2min; Capping DMAP, acetic anhydride, sym-collidine / 30s; Oxidation iodine / 12s. The β-cyanoethyl and the base protecting groups were deprotected with 50/50 v / v CH ₃ NH ₂ / NH ₃ at 65 ° C. in 7 min. After that, rapid cooling and washing out is necessary to stop the reaction. There is no attack on the surface, which leads to the oligonucleotide being split off from the support, although Si-O bonds are base-labile, which is due to the fact that the exposure time of the strongly basic reagents is kept very short. No additional "spacer" is required. The coupling of the deoxyphosphoamidites provided with protective groups takes place directly on the NH ₂ groups. The particles loaded with oligonucleotide were then washed three times with water and twice with acetonitrile and air-dried.

By the method of "split-couple-combine" is one carrier particle each loaded with a sequence if you have a synthesis with full yield presupposes. full Yield is only an idealization and never achieved in reality, but carrier mentioned above give good results in this regard.

Since the number of oligonucleotides in a library with the length n of the oligonucleotide rises sharply with 4 ⁿ , it does not make sense for an application to produce libraries longer than 8 or 9 mers, since there are not enough particles of the carrier material per synthesis to include all library representatives.

This makes it possible to "screen" the particles with a surface-bound oligonucleotide with proteins as a "target" in ~ 1μM protein solution in the buffered system if they have previously been blocked with a "blocking" solution of 1% bovine serum albumin solution in a so-called " selection "buffer (20mM Tris-acetate, 140mM NaCl, 5mM KCl, 1mM MgCl ₂ , 1mM CaCl ₂ , pH = 7.4) were incubated at 37 ° C for 1h. The serum albumin binds to the polar carrier surface so that it is no longer accessible to the fluorescent-labeled protein, since otherwise all particles would become fluorescent. However, this measure does not prevent the specific interactions between the protein and the oligonucleotide bound on the surface.

There is no excessively strong fluorescence of the background, so that screening with fluorescein isothiocyanate-labeled proteins is possible in 20mM Tris-acetate, 140mM NaCl, 1mM CaCl ₂ , 5mM KCl, 1mM MgCl ₂ , pH = 7.4.

Hybridization experiments could be carried out with good results in the same buffer system, the “sense” sequence bound to the support combined with the fluorescent labeled “antisense” sequence leading to a strong fluorescence of the particles. In the "scrambled" sequence, however, the particles remain dark. Positive hits on "Sceening" stand out well from the background if the concentration of the selected "target" is not increased too much, since otherwise the background fluorescence and also the non-specific binding to the support become too strong.

In 4 and 5 the superficially bound sequence is: 5'-GTCCCTGTTCGGGCGCCATTTT-3 ', incubated with 3'-fluorescent labeled sequence 5'-TGGCGCCCGAACAGGGAC-3' (1.45 μM) ( 4 ), incubated with 3'-fluorescent labeled sequence 5'-CGGACCTGGCAAGGGACC-3 '(1.09 μM) ( 5 ).

On Cleavage of the P-N bond with 80% acetic acid is possible in 30 min at RT the oligonucleotide of a single particle in solution to get.

Now sequencing or further use of the oligonucleotide can be connected since enough oligonucleotide (~ 11-14 μmol / g carrier) is available on a particle for MALDI-MS or PCR. This means that a few picomoles of oligonucleotide per particle are removed. By means of radio labeling (e.g. γ- ³² P-ATP), the purity of the oligonucleotide on a particle can be documented very nicely. Due to the strongly basic conditions, there can still be a large loss of substance when deprotecting the oligonucleotide on the support, but the amount remaining on the solid phase is sufficient to supply sufficient substance for the analytical methods used.

This is shown by the MALDI spectra of individual carrier particles of the sequence 5'-ACATCAAGTTT-3 'on CPG triazine NH ₂ ( 6 ) and CPG NH ₂ ( 7 ).

The peak at 3317.3 Da ( 6 ) the 3'-OH oligonucleotide, the peak at 3395.34 Da ( 7 ) the 3'-phosphorylated oligonucleotide. Such 3'-phosphorylated products logically result from the cleavage of the PN bond, according to SM Gryaznov, RL Letsinger. The matrix used consists of ATT in 50/50 water / acetonitrile with the addition of 12 mM spermine. For each "spot", 1μl sample, then 1μl of the matrix solution were used for MALDI.

The settings used shows 8th ,

9 shows an electropherogram (20% analytical gel, 19/1 acrylamide / bisacrylamide) of three individual particles of an 8-mer library with TTTT spacer on CPG NH ₂ , compared with a sequence 5'-ACATCAAGTTT-3 '(band rightmost).

The samples were radiolabeled with γ- ³² P-ATP. For this, 1/5 to 1/10 of a particle was used. This allows one and the same particle to be examined for purity of the oligonucleotide before use.

there It should be borne in mind that there are no cleaning steps on the substance Individual particles possible are. shortened Sequences are recognizable because of the gel electrophoresis with radiolabelling represents a very sensitive method, the purity of oligo or to recognize polynucleotides.

there it should be noted that not every carrier particle receives a signal is, especially when it comes to particles from a library, since the loading of the particles can fluctuate. But even the fluctuations the amount of substance is opposite the carriers used so far reduced when using this combination of methods.

A potential Using the combination of methods shown is to find it of oligonucleotide sequences by "screening" oligonucleotide libraries which to a selected "target", such as a protein or bind a low molecular weight, organic molecule with high affinity. Some such aptamers are found in M. Famulok, G. Mayer and M. Blind, Acc. Chem. Res. (2000), 33, No. 9, 591-599.

Claims (6)

Process for the production of nucleotides bound on porous glass (CPG) with the following steps: a) functionalizing the CPG surface with NH ₂ groups, b) coupling the deoxyphosphoamitide provided with protective groups directly onto the NH ₂ groups and building up the nucleotide, c ) Splitting off the protective groups with a solution containing aminoalkane and / or a mixture of an aminoalkane and a base at room temperature to 100 ° C. for 2 to 20 min. A method according to claim 1, characterized in that a CH ₃ NH ₂ / NH ₃ mixture of 50:50 v / v is used. A method according to claim 1 or 2, characterized in that that the temperature is in the range of 45 to 80 ° C, preferably 60 to 70 ° C. Method according to at least one of claims 1 to 3, characterized in that the splitting off during a period of 4 to 8 min., preferably 5.5 to 7.5 min. is carried out. Method according to at least one of the claims che 1 to 4, characterized in that phosphoamitides with acetyl protective group are used. Nucleotide library can be produced by a method according to at least one of the claims 1 to 5.