RU2520094C1 - Combinatorial method for obtaining dna-aptamer thrombin inhibitors and aptamer oligonucleotides (versions) - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method for modular design of DNA aptamers capable of specific and high-affinity binding of thrombin, which have stabilised basic substructure, provides for assembly of their structure by modelling using a combination of three structural modules containing a quadruplex nucleic acid module, a duplex nucleic acid module and a nucleic acid module that binds them and has a non-canonical structure by determining tertiary structure with a spectral circular dichroism method with confirmation of the fact of formation of more stable G-quadruplex that is different from quadruplex structure of initial structural quadruplex module.

EFFECT: application of a modular design method allows improving assembly efficiency of antithrombin DNA-aptamers having increased stability under physiological conditions.

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Description

The invention relates to the field of biochemistry and medicine, specifically to a method for producing new aptameric inhibitors based on a combination of structural modules of known aptamers.

The invention relates to the field of biotechnology and medicine, specifically to a method for stabilizing antithrombin DNA aptamers and obtaining new stable antithrombin DNA aptamers, which can serve as the basis for the development of medicinal anticoagulant and antithrombotic drugs.

Thrombin is an enzyme of the hydrolase class, an essential component of the coagulation system of human and animal blood. Prothrombin is present in the blood as an inactive precursor and is activated by prothrombinase (active thromboplastin). By its chemical nature, thrombin is a glycoprotein with a molecular weight of about 40,000; contains about 5% carbohydrates. Thrombin belongs to the class of serine proteinases (trypsin, etc.). The thrombin molecule consists of two polypeptide chains connected by a disulfide bond: A-chain and B-chain, which has an active center of the enzyme and a carbohydrate component. Thrombin exists in several active forms, which differ in the structure of the B-chain. The main function of thrombin is the conversion of fibrinogen to fibrin. Thrombin hydrolyzes four arginine-glycine bonds in the fibrinogen molecule; in this case, four peptides are cleaved off and a fibrin monomer is formed, which then aggregates into a fibrin clot, which is the basis of the thrombus. The limited proteolysis reactions involving thrombin also accompany the activation of factor XIII (fibrin-stabilizing factor) and factors V and VIII, which are involved in reactions of the internal mechanism of blood coagulation. With the participation of thrombin, platelet aggregation occurs, as well as compression (retraction) of a blood clot. It was shown that the relative excess of thrombin in the body reflexively activates the so-called. anticoagulant system, while heparin and plasminogen activator enter the bloodstream, which are involved in maintaining the liquid state of the blood.

Thrombin is inactivated by diisopropyl fluorophosphate, which blocks the hydroxyl group of serine, which is part of the active center, and other inhibitors characteristic of the serine proteinase group. In blood, thrombin is inactivated by plasma antithrombin: a2-macroglobulin, antithrombin III and (or) heparin. A specific non-plasma thrombin inhibitor is the hirudin polypeptide found in the salivary glands of a medical leech.

In recent years, a new type of thrombin inhibitor, DNA aptamers, has been developed.

Aptamers are small nucleic acid molecules that can serve as highly specific receptors for any target, from whole cells to low molecular weight organic compounds. Oligonucleotide aptamers with the desired properties are isolated from random sequence libraries by in vitro selection methods (SELEX), using their ability to specifically interact with corresponding immobilized targets / ligands.

So, one of the first DNA aptamers that inhibit thrombin was found by Schultze P. et al. "Three-dimensional solution structure of the thrombin-binding DNA aptamer d (GGTTGGTGTGGTTGG)", J. Mol. Biol. 1994 Feb 4; 235 (5): 1532-47. The DNA oligonucleotide dGGTTGGTGTGGTTGG, a thrombin-binding aptamer, specifically binds to thrombin and inhibits enzyme activity in the chain of reactions leading to blood coagulation. The spatial structure of this thrombin-binding aptamer may be the basis for the development of improved thrombin inhibitors with similar structural motifs.

In Nagatoishi (Nagatoishi S. et al., "Circular dichroism spectra demonstrate formation of the thrombin-binding DNA aptamer G-quadruplex under stabilizing-cation-deficient conditions", Biochem Biophys Res Commun. 2007 Jan 19; 352 (3): 812-7. Epub 2006 Nov 27) describes an antithrombin aptamer having a G-quadruplex structure.

Gatto B. (Gatto B. et al., "Nucleic acid aptamers based on the G-quadruplex structure: therapeutic and diagnostic potential", Curr Med Chem. 2009; 16 (10): 1248-65) describes a G-quadruplex antithrombin aptamer , and also reviews current knowledge about aptamers based on G-quadruplex structures, as well as evaluates their diagnostic and therapeutic potential (as biotechnological preparations) for the detection and treatment of severe pathologies, including vascular, cancer and viral diseases.

In addition, aptamer DNA was obtained for thrombin, the biomedical properties of which are described, for example, in a number of publications:

- IE 920561 A1 (GILEAD SCIENCES) dated 08/26/1992, which describes an aptamer oligonucleotide that specifically binds to thrombin and has a GGXTGG nucleotide sequence, where X is T, A, U, dU or G, in particular contains a nucleotide sequence GGTTGGTGTGGTTGG.

- US 2005176940 A1 (UNISEARH LTD) dated 09/11/2005, which describes an aptamer oligonucleotide that specifically binds to thrombin and has a GGTMGGXGGTTGG nucleotide sequence, where M is A or T, and X is a sequence from 2 to 5 of any nucleotides or their modifications.

- Griffin L.C. et al., "In vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits", Blood. 1993 Jun 15; 81 (12): 3271-6, describes an aptamer oligonucleotide having the sequence GGTTGGTGTGGTTGG.

However, a common drawback inherent in DNA aptamers that bind to thrombin is their low stability in vivo.

Aptamer DNA dGGTTGGTGTGGTTGG (15TGT), specifically binding thrombin, was first obtained by SELEX (Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6 ; 355 (6360): 564-6). 15TGT, a 15-link thrombin-binding DNA aptamer, was able to inhibit thrombin. In contrast to the currently widely used thrombin inhibitors, such as heparin, 15TGT has low immunogenicity and short life time in the body, it is a promising object for the production of drugs. 15TGT has an intramolecular so-called G-quadruplex structure formed by two stacked G-quartets connected by one TGT loop and two TT loops.

Stacking interactions (van der Waals pi-electron hydrophobic interactions by nature) lead to the fact that flat aromatic systems of heterocyclic bases self-organize into stack-like structures with an interplanar distance of 3.4-3.8 angstroms. In this case, full or partial overlap of the base planes occurs.

The 15TGT G-quartets are composed of four guanines joined non-covalently via Hugsteen hydrogen bonds. The union of two G-quartets is called a G-quadruplex. G-quadruplexes can be formed from two or four DNA molecules, giving intermolecular complexes. G-quadruplexes have a characteristic spectrum of circular dichroism.

The 15TGT antithrombin aptamer is a minimal monomolecular quadruplex. In solution, 15TGT loses the structure of the quadruplex already at 25 ° C, which is much lower than physiological temperatures.

From RU 2401306, 10/10/2010, an aptamer oligonucleotide direct thrombin inhibitor is known, in particular an original 31-link aptamer DNA, code-named RE31 and having the sequence GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC. This aptamer is not inferior in its ability to inhibit the activity of thrombin to the 31-unit aptamer 31ТВА (TBA-thrombin binding aptamer) with the sequence CACTGGTAGGTTGGTGTGGTTGGGGCCAGTG, one of the most highly effective antithrombin aptamers described above.

From RU 2429293, 09/20/2011, DNA aptamers that inhibit thrombin and a method for stabilizing their structure are known.

The method involves the formation of an additional system of stacking interactions using heterocycles or their analogues by increasing the surface of the aromatic system of heterocycles or their analogues, due to the application of methods for determining the tertiary structure or molecular modeling with confirmation of the formation of contact of the aromatic system of heterocyclic bases or their analogues with a quartet of G-quadruplex which is adjacent to the lateral loop. The presented method allows to increase the assembly efficiency of antithrombin DNA aptamers and to increase the stability of their structure under physiological conditions.

In this known manner, the aptamer oligonucleotides DTI-G84, DTI-P91, DTI-A6, DTI-r91, DTI-Fx7 were obtained.

In particular, the aptamer oligonucleotide DTI-G84 is an oligodeoxyribonucleotide containing the nucleotide sequence

Nx-GGGTTGGGTGTGGGNTGGGNy,

where N is any nucleotide from the series G, A, T, C, and x and y are any number of nucleotides that are not equal to each other and which specifically and highly affinity binds to thrombin.

However, DNA aptamers against thrombin are known to have a very short (not more than a few minutes) lifetime in the bloodstream [Griffin L.C., Tidmarsh G.F., Bock L.C., Toole J.J., Leung L.L.K. Blood 1993, v. 12, p. 3271-3276]. This property can prevent the creation of effective drugs on their basis. In this regard, to extend the lifetime in vivo base oligonucleotide sequences can undergo chemical modifications. These modifications, firstly, protect aptamers from the action of nucleases, and secondly, slow down their excretion through the kidneys. Modified aptamers with potential antithrombotic properties are known against the von Willebrand factor [Gilbert J.C., De Feo-Fraulini T., Hutabarat R.M., Horvath C.J., Merlino P.C., Marsh H.N. et al. Circulation 2007, v. 116 pp. 2678-2686] and factor IX [Dyke C.K., Steinhubl S.R., Kleiman N.S., Cannon R.O., Aberle L.G., Lin M. et. al. Circulation 2006, v.114, pp.2490-2497].

Thus, the technical task of the present invention is to develop a combinatorial method for constructing antithrombin DNA aptamers based on three classes of known structural modules with improved stability of its structure.

The stated technical problem is achieved by the claimed group of inventions, which includes a method for producing a DNA aptamer having antithrombotic activity, and aptamer oligonucleotides Num1, Num6, Num7 obtained by the above method and representing oligodeoxyribonucleotides containing certain nucleotide sequences of the general formula (I), ( II) and (III).

So, the stated technical problem is achieved by the method of producing a DNA aptamer with antithrombotic activity and having a stable (stable) main substructure obtained by assembling the structure by modeling it from a combination of three structural modules (combinatorial method) containing a quadruplex nucleic acid module, a duplex nucleic acid module (duplex) and the nucleic acid module connecting them, having a noncanonical structure, by determining the tertiary structure of its spec eral by circular dichroism confirming the fact of the formation of a stable G-quadruplex different from the quadruplex source module quadruplex structure.

Other inventions of the claimed group are aptamer oligonucleotides (variants) obtained by the above method.

So, another invention of the claimed group is the aptamer oligonucleotide Num1 having antithrombotic activity, which contains a stabilized G-quadruplex structure and is an oligodeoxyribonucleotide containing the nucleotide sequence of the general formula:

N _x CACTGGGGGGTTGGTGTGGTTGGATCCAGTGN _y (I),

where N denotes any nucleotide selected from A, C, T, G; the subscripts x and y denote any number of nucleotides.

The invention of the claimed group is also an aptamer oligonucleotide Num6 having antithrombotic activity, which contains a stabilized G-quadruplex structure and is an oligodeoxyribonucleotide containing a nucleotide sequence of the general formula:

N _x CGCCTAGGTTGGTGTGGTTGGGGGGCGN _y (II),

where N is any nucleotide from the series G, A, T, C, and x and y are any number of nucleotides that are not equal to each other, and which specifically and highly affinity binds to thrombin.

Another invention of the claimed group is the aptamer oligonucleotide Num7 having antithrombotic activity, which contains a stabilized G-quadruplex structure and is an oligodeoxyribonucleotide containing the nucleotide sequence of the general formula:

N _x GTGACCTAGGTTGGTGTGGTTGGGGGGTCACN _y (III),

where N is any nucleotide from the series G, A, T, C, and x and y are any number of nucleotides, and which specifically and highly affinity binds to thrombin.

The claimed invention describes the development of a combinatorial design of the structures of DNA aptamers with the following primary structure:

three-module DNA aptamer (Num1)

N _x CACTGGGGGGTTGGTGTGGTTGGATCCAGTGN _y (I),

three-module DNA aptamer (Num6)

N _x CGCCTAGGTTGGTGTGGTTGGGGGGCGN _y (II),

three-module DNA aptamer (Num7) N _x GTGACCTAGGTTGGTGTGGTTGGGGGGTCACN _y (III),

where N is any nucleotide selected from A, C, T, G; the subscripts x, y indicate the number of nucleotides.

1) The value of X is limited by the total weight of the molecule, at which the nature of its pharmacokinetics is likely to change, i.e. total weight up to 20 kDa, respectively, Xmax = 15.

2) Aptamer (Num1) - structural module No. 1 of aptamer 31TGT, structural module

No. 2 of aptamer 31TGT and structural module No. 3 of aptamer RE31;

- aptamer (Num6) - structural module No. 1 of the aptamer 31TGT, structural module No. 2 of the aptamer Nu172 and structural module No. 3 of the aptamer 31TGT;

- aptamer (Num7) - structural module No. 1 of aptamer 31TGT, structural module No. 2 of aptamer RE31 and structural module No. 3 of aptamer RE31.

When implementing the inventive method, known quadruplexes and duplexes, for example, such as duplexes and quadruplexes of the RE31, 31TGT and Nu172 aptamers described and known from sources such as RU 2401306, 10.10.2006 [31-link DNA aptamer with code name RE31]; 31-link aptamer 31TGT (RU 2429293, 09/20/2011) and 20090221680, 09/03/2009 [26-link DNA aptamer, code-named ARC2172].

Moreover, as the module connecting the quadruplex module and the duplex module, the parts of the above aptamers (RE31, 31TGT and Nu172) having a noncanonical structure and located between the mutually complementary flanking sequences forming the duplex and the quadruplex pattern are used in the method.

Thus, the combination of modules 2 and 3 of the RE31 aptamer increases the stability of the aptamer by 5 ° C, and also removes the commercially diverse variety. According to some reports, the use of a shorter duplex module in combination with module 3 of the Nu172 aptamer improves binding to the target molecule in a clean system, without the presence of extraneous proteins.

Thus, the aptamer was constructed by combining modules of types 1, 2, and 3, which would surpass previously known aptamers in such parameters as homogeneity of structural forms and stability.

So, the technical problem is solved by searching for structural factors of stabilization of G-quadruplexes of antithrombin DNA aptamers by circular dichroism spectroscopy. Based on the identified factors, a rational design of modified aptamers is carried out. Direct chemical synthesis of modified stable structures is carried out, their thermal stability is compared with previously known aptamers.

So, the technical result of the claimed group of inventions is to increase the efficiency of the assembly of antithrombin DNA aptamers and increase the stability of their structure under physiological conditions. Factors determining the positive effects for stabilization of antithrombin DNA aptamers can be used for other G-quadruplex structures, including aptamers for other targets and for telomeric structures.

The invention describes the development of a combinatorial design of DNA aptamer structures with the following primary structure:

- three-module DNA aptamer N _x CACTGGGGGGTTGGTGTGGTTGGATCCAGTGN _y (I),

- three-module DNA aptamer N _x CGCCTAGGTTGGTGTGGTTGGGGGGGCGN _y (II),

- three-module DNA aptamer N _x GTGACCTAGGTTGGTGTGGTTGGGGGGTCACN _y (III),

where N is any nucleotide selected from A, C, T, G; the subscripts x, y indicate the number of nucleotides.

The corresponding oligodeoxyribonucleotides were synthesized by standard solid phase synthesis. For all aptamers, the thermal stability of the G-quadruplex structural module is determined.

Brief Description of the Drawings

Figure 1. Schematic representation of the spatial structure of the aptamer 31TGT described in the literature.

Figure 2. Circular dichroism spectra (dependence of molar ellipticity on wavelength) of aptamer oligonucleotides that are stabilized according to patented principles (20 mM Tris-HCl buffer, pH = 7.5, 140 mM NaCl, 5 mM KCl, in the temperature range from 5 ° C to 80 ° C in increments of 5 ° C): A) aptamer Num1. B) aptamer Num6. C) aptamer Num7.

Figure 3. Melting curves (temperature dependence of molar ellipticity at a wavelength of 294 nm) obtained on the basis of circular dichroism data of aptamer oligonucleotides, which are stabilized according to patented principles. A) aptamer Num1. B) aptamer Num6. C) aptamer Num7.

Description of the invention

According to preliminary X-ray diffraction analysis of the complex of the aptamer with thrombin from the side of the aptamer, the lateral TT loops participate in the complex, the conformation of which is determined by the structure of the G-quadruplex. In this regard, any effect or modification that leads to stabilization of the quadruplex structure affects the interaction of the loops with thrombin. The applicant has established that, when a combination of structural modules occurs during the formation of the sequence G _n (where N> 5), it leads to stabilization of the aptamer structure due to stabilization of the quadruplex. Quadruplex stabilization can occur for two reasons. Firstly, due to an increase in stacking interactions as such, including the effect of screening of heterocyclic bases of the upper quartet from water. Secondly, the introduction of the poly-G sequence shifts the equilibrium towards the formation of multimolecular quadruplex structures, which are much more stable than monomolecular ones.

Detailed Description of Preferred Embodiments

The following examples set forth the preferred materials and methods of the present invention.

Example 1

The synthesis of DNA aptamers according to the invention was carried out according to a standard method on a solid-phase support (Oligonucleotide Synthesis, M.J. Gait ed, 1984; Sambrook, Fntsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989)).

The synthesis was carried out by the solid-phase method on an Applied BioSystems 380b automatic synthesizer using standard nucleotide derivatives. As a polymer carrier, CPG glass balls (control pore glass) with pore sizes of 500 and 1000 Å were used. The first nucleoside with a 3'-hydroxyl group was attached to the carboxyl group of the modified polymer carrier. Each synthesis cycle consisted of four stages:

- detritification to remove the dimethoxytrityl group by treatment with trichloroacetic acid and liberate the 5'-OH group;

the addition of a nucleotide in the form of β-cyanethyl-N, N-bis-diisopropylamidophosphite nucleoside;

- the β-cyanethyl group is removed after completion of the synthesis by treatment with ammonia. The activating reagent during condensation is tetrazole;

- caching. The addition of amidophosphite at the condensation stage does not occur quantitatively; therefore, about 2% of the 5'-OH groups do not react. At subsequent stages of the synthesis, nucleotides can be attached to them, which ultimately leads to gaps in the nucleotide sequence. In this regard, it is necessary to protect free 5'-OH groups, i.e. capping by reaction of acetic anhydride in the presence of N-methylimidazole;

- oxidation of phosphite ether to phosphate iodine in pyridine.

Example 2

A variant of the antithrombin aptamer obtained by K. Ikebukuro et al., Inhibited thrombin better than 15TGT, while its structure had additional nucleotides from both ends of the molecule. The spatial structure of the 31-link 31TGT aptamer was described by circular dichroism spectroscopy methods: the ends of the molecule are closed in a double-stranded structural module and connected to the quadruplex module by internal loops of two nucleotides each, the so-called connecting structural module (Figure 1). Aptameric inhibitors of RE31 and Nu172 are thought to have a similar modular structural organization.

According to the patented principle of structure stabilization due to the formation of strong stacking interactions, the connecting structural module No. 3, belonging to

31TGT [TA \\ GG], was replaced by the corresponding module RE31 [GG \\ AT], so the combination of structural modules [module No. 1 - 31TGT, module No. 2 - 31TGT, module No. 3 - RE31] leads to the appearance in the primary the structure of the sequence G _n (where N> 5). The guanine residue forms more stable stacking interactions and has more potential for the formation of hydrogen bonds. The oligonucleotide sequences obtained by the modular design were investigated by the circular dichroism method. Thermal stability was investigated in the wavelength range from 220 to 350 nm with a temperature variation from 5 ° C to 80 ° C in increments of 5 ° C. In the observed temperature range, a significant change in the characteristic maximum of circular dichroism at 294 nm occurs, which allows us to evaluate the thermal stability of the G-quadruplex structure. The structure of the new aptamer [module No. 1 - 31TGT, module No. 2 - 31TGT, module No. 3 - RE31] showed increased stability (Figa). A change in the general appearance of the spectrum (Fig. 2A) compared with the standard spectrum of circular dichroism of antiparallel monomolecular G-quadruplex indicates a shift in equilibrium towards the formation of intermolecular G-quadruplex structures, which leads to an overall increase in the stability of the aptamer.

The modified aptamer with the sequence N _x CACTGGGGGGTTGGTGTGGTTGGATCCAGTGN _y was assigned the index Num1.

Similarly, by combinatorial design, Num6 (2B and 3B) and Num7 aptamers (2B and 3B) were obtained, the nucleotide sequences of which were previously disclosed.

Example 3

The discussion of the results

It was shown that the Num6 aptamer has a higher thermal stability of the G-quadruplex module compared to previously known aptamers. Similar effects are observed for all combinations of structural modules leading to the formation of the G ₆ sequence in the primary aptamer sequence.

By combinatorial design, aptamer thrombin inhibitors having higher thermal stability than previously described in the literature have been successfully obtained. The structure and properties of modular aptamers are modeled on the basis of circular dichroism spectroscopy data and are given in the corresponding examples illustrating, together with graphic material, obtaining a stable DNA aptamer using the combinatorial method.

Claims (4)

1. A method for producing a DNA aptamer having antithrombotic activity and having a stable basic substructure by assembling its structure by modeling from a combination of three structural modules containing a quadruplex nucleic acid module, a duplex nucleic acid module and a nucleic acid module connecting them having a non-canonical structure, by determining the tertiary structure by its spectral method of circular dichroism with confirmation of the formation of a more stable G-quadruplex, different from the quadruplex structure of the original structural quadruplex module. 2. Aptamer oligonucleotide Num1 having antithrombotic activity, which contains a stabilized G-quadruplex substructure obtained by the method according to claim 1, which is an oligodeoxyribonucleotide containing a nucleotide sequence of the general formula:
N _x CACTGGGGGGTTGGTGTGGTTGGATCCAGTGN _y (I),
where N denotes any nucleotide selected from A, C, T, G; the subscripts x and y denote any number of nucleotides. 3. Aptamer oligonucleotide Num6 having antithrombotic activity, which contains a stabilized substructure obtained by the method according to claim 1, which is an oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
N _x CGCCTAGGTTGGTGTGGTTGGGGGGCGN _y (II),
where N is any nucleotide from the series G, A, T, C, and x and y are any number of nucleotides that are not equal to each other, and which specifically and highly affinity binds to thrombin. 4. Aptamer oligonucleotide Num7 having antithrombotic activity, which contains a stabilized substructure obtained by the method according to claim 1, which is an oligodeoxyribonucleotide containing the nucleotide sequence of the General formula:
N _x GTGACCTAGGTTGGTGTGGTTGGGGGGTCACN _y (III),
where N is any nucleotide from the series G, A, T, C, and x and y are any number of nucleotides, and which specifically and highly affinity binds to thrombin.

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