Method of producing artificial oligonucleotides potentially capable of forming imperfect g-quadruplexes. RU patent 2509802.

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically a method of producing artificial oligonucleotides that are potentially capable of forming non-canonical structures that stable in physiological conditions and conditions close to physiological, said structures being imperfect G-quadruplexes (lmGQ) which include one nucleotide substitution in the G₄ plane in the G-quadruplexes (GQ). Said method includes using an algorithm describing nucleotide sequences in form of a defined set of formulae for further synthesis of selected oligonucleotides.

EFFECT: invention enables to use bioinformation analysis to obtain artificial oligonucleotides that are potentially capable of forming a new conformation - imperfect G-quadruplexes.

4 dwg, 2 tbl, 2 ex

The technical field to which the invention relates

The invention relates to the field of genomics and structural biology, and can be used for analysis of genomic texts, study of structural-functional properties and mechanisms of DNA and RNA. Prediction of the existence of new non-canonical tertiary structures of polynucleotides, namely imperfect G-QUADRUPLEX (ImGQ), potentially stable in physiological conditions, will allow to find the exact molecular targets and biomarkers of pathological human States, to develop new drugs directed action. The subject of this patent search algorithm ImGQ, potentially stable in physiological conditions (1), and the method for the oligonucleotides capable of forming ImGQ (2).

The level of a technical condition

In recent years shows that the native DNA in addition to the prevailing B-form there are other non-canonical space structure (conformers, ncDNA). Such sites are considered as structural-mediated controls genomes, such as regulators of gene transcription [1]. Among the famous ncDNA can be called G-QUADRUPLEX (GQ) and I-motives [2, 3], triplexes and fragments of parallel duplexes [4-6]. The most studied of ncDNA G-QUADRUPLEX.

Intramolecular G-QUADRUPLEX (eng. G-quadruplex, and G-tetrads, GQ) - oligonucleotide or fragment polinucleari acid that can form chetyrekhzvezdochnuyu spiral, stable interaction of four Gurinovich grounds. Each G-Quartet fastened in the amount of eight hydrogen bonds and forms a flat structure (Figure 1).

GQ found in vivo in the promoters of human genes c-kit, c-myc, hTERT and others [7-12]. The sequence of nucleotides, are able to form QG, widely represented in noncoding 5'-region of many genes and can be directly involved in the regulation of their expression [13]. Education GQ tend many micro and minisatellite repetitions of the human genome, for example, telomeric repeat (TTAGGG) n or satellite (GGGTCT) n . It is shown that mutations in the field of GQ are the basis of many diseases [14]. Currently GQ-sites of polynucleotides are considered as a promising therapeutic target in Oncology [15].

It is known that the formation of GQ you want the movie polynucleotide meet certain requirements. The sequence potentially capable of forming GQ, must comply with the following formula (G x L yi )G x , where x=2, 3, 4... - number of G-quartets GQ; y=1, 2, 3... - the number of nucleotides in the i-th loop connecting 4 G plane.

To search PQS (Potential Quadruplex Sequence) in fragments polynucleotides and genomic texts has been developed algorithm [5, 16] (the closest prototype) and created available online program, for example, QGRS (http://bioinformatics.ramapo.edu/QGRS/analyze.php) and quadfinder (http://miracle.igib.res.in/quadfinder/).

To confirm the existence of every found GQ must obtain the appropriate oligonucleotide and determine its conformation instrumental and physical-chemical methods.

In this application we first prove that G-QUADRUPLEX not describe all of the conformational state of polynucleotides, stable stacking interaction four Gurinovich grounds. Imperfect G-QUADRUPLEX (eng. ImG quadruplex, and also ImG tetrads, ImGQ) - oligonucleotide or fragment polinucleari acid, similar to GQ sequence, but bearing in contrast to GQ, one replacement of the Quartet-forming guanine to another nucleotide or equivalent (Figure 1A-D), can also have stable conformation in natural conditions.

None of the known algorithms for analysis primary DNA sequences on the basis of the education potential of the non-canonical conformations does not take into account the rules of formation of imperfect quadruplexes, and search programs, created on their basis, do not enable identification of sites capable of forming ImGQ.

Disclosure of inventions and the technical result

The technical result of the invention is to create a new instrument bioinformatics analysis that identifies the primary sequences polynucleotides sites, can form potentially stable in physiological terms, new conformation - imperfect G-QUADRUPLEX (ImGQ). To achieve that it was necessary to develop a method of obtaining ImGQ-oligonucleotides natural and model sequences and their examples to prove the existence ImGQ and confirm their high stability (see data in table 2).

The technical result is achieved by the fact that on the basis of the study of properties of oligonucleotides and literature data generated rules ImGQ (algorithm).

The proposed algorithm of search ImGQ provides for the description of sequences of potential imperfect quadruplexes set of formulas that can be represented in the form of a table (table 1) format k x 4, where n (the number of rows corresponds to the number of quartets formed four consecutive parts of the site and may not be less than 4 (k = 4). The sum of the items in the table describes all monopsonists ImGQ, able to receive conformation ImGQ in physiological and near-physiological conditions (for example, in 50-100 mM K+, Cl, 10 mm TrisHCl, pH 7-8).

It is important to note that in this approach, described earlier GQ (committed GQ) become a special case of ImGQ realized at N=G.

To justify the proposed algorithm that seeks to create a stable conformers, it is necessary to clarify a number of key positions.

1. The possibility of forming only intramolecular GQ.

In vivo concentrations polynucleotides relatively small, in addition molecules DNA and RNA are part of a complex supramolecular complexes with various biopolymers and low-molecular compounds, which in most cases prevents the formation of intermolecular GQ.

2. ImGQ, stable in physiological conditions that may emerge in the case of four or more Quartet GQ bearing unit replacement in G 4 planes.

It is known that the nucleotide substitutions in G 4 planes and even change the nature of the chemical bond between guanine destabilize two and trequartista GQ [16-19]. Example 1 illustrates the effects on the stability chetyrehspalnyh GQ - gGQ (sequence 1) and Ctg (sequence 2) (table 2) - the one - and two-nucleotide substitutions G to other nucleotides in QUADRUPLEX-forming units GGGG. From the Table 2 data shows that a single replacement lead to a slight reduction of the melting temperature GQ (up to 82°C, for bclGQ (sequence 3) and to 62-82 C for oligonucleotides Ct series). Note that the stability of these structures ImGQ remains high and considerably exceeds the physiological norm.

Replacement of two guanine in G4-plane reduce the stability of the tertiary structure of oligonucleotide and the erosion of the spectra KD (table 1. Figure 2A, oligomers aGQ (G/A G/A) (sequence 4) and tGQ (G/A,G/T) (sequence 5)). So, for example, melting point tGQ (sequence 5) does not exceed 52°C, therefore it cannot be reasonably attributed to stable in physiological terms, GQ, and oligomer aGQ (sequence 4) forms of molecular structure.

Table 1.

Formula sequences of fragments of polynucleotides (PImQS), can potentially take a conformation imperfect quadruplexes.

A number of

Sequence potential imperfect ImGQ

NG k-1 (L x G k ) 3

G k L NG x k-1 (L x G k ) 2

(G k L x ) 2 NG k-1 L x G k

(G k L x ) 3 NG k-1

GNG k-2 (L x G k ) 3

G k L x GNG k-2 (L x G k ) 2

(G k L x ) 2 GNG k-2 L x G k

(G k L x ) 3 GNG k-2

G 2 NG k-3 (L x G k ) 3

G k L x G 2 NG k-3 (L x G k ) 2

(G k L x ) 2 G 2 NG k-3 L x G k

(G k L x ) 3 2 G NG k-3

G k-1 N(L x G k ) 3

G k L x G k-1 N(L x G k ) 2

(G k L x ) 2 G k-1 N(L x G k )

(G k L x ) 3 G k-1 N

where k is the number of quartets formed four consecutive G-rich snippets sequence (k = 4); L (1 to 7) - any nucleotide in the loop GQ; N is any nucleotide in the Quartet GQ. If N=G, the formula describes a perfect GQ.

About the formation of chetyrehjadernogo a highly imperfect QUADRUPLEX evidenced by the fact that the stability ImQG bclGQ (G/A) (sequence 3) - the natural sequence of the promoter region of the gene BCL-2 - significantly (to 27 degrees Celsius (C) exceeds stability trekhsvetnogo homolog trGQ (sequence 6), the formation of which was predicted using previous search algorithm QG (for example, the program quadfinder, based on known algorithm [16]).

Nature KD-spectra ImGQ (Figure 2A and D) in the presence of potassium ions, stabilizing GQ, and differential melting curves ImGQ (Figure 2B) typical for GQ. Moreover, ImGQ as perfect QUADRUPLEX (in this example, Ctg (sequence 2)) lose stability conformation in the presence of lithium ions (Figure 2D).

Reliable evidence of the formation ImGQ is the comparison of the spectra of TMR (area 12 ppm) natural ImQG Ct1 (sequence 7) and the spectrum of the oligonucleotide GQ1 (sequence 8). Both sequences in accordance with algorithm of search PQS able to form 2-tetrad GQ because the maximum length of four G-units in their composition is equal to two. This means that in the field Justiniskiu interactions (region 12 ppm) should be 8 signal that corresponds to the range of GQ1 (sequence 8) (Figure 3, bottom). However, in the spectrum of oligomer Ct1 (sequence 7) (Figure 3, top range) not found 8 and 14 proton signals involved in education planes ImGQ patterns. This fact is well described chetyrehmetrovy scheme structural ImGQ (Figure 3).

The above argument leads to the conclusion that the new search algorithm is highly stable ImGQ should relate to four and more Quartet structures ImGQ and faithful for n > 4.

The length of loops ImQG by analogy with GQ is considered in the range from 1 to 7 of nucleotides [20]. The composition of the loops can enter any nucleotide.

The interval 1-7 is not mandatory and can be extended. However, the limitation of the length of loops to 7 links increases the probability of detection of the most stable in physiological conditions GQ [20, 21], this pattern can be attributed to ImGQ.

3. The algorithm provides for the search of potential ImGQ that can exist simultaneously, i.e., for example, block GGGTGG when searching chetyrehspalnyh ImGQ counted as one, not as three fragments of different GQ resulting from alternative folding.

Taking into account the above rules developed a computer program to find ImGQ in genomic nucleotide data (table 1), named ImGQfinder (filing date of an application for the state registration: 06.09.2012). It can help analyze large volumes of data, to get a list of sequences ImGQ indicating the position in the genome, statistics occurrences of defects (N) and other results. The program is written in C#. The output format can be changed.

The analysis of primary nucleotide sequences of the direct and inverse chains 18 human chromosome (http://www.ncbi.nlm.nih.gov/nuccore/NC_000018.9) using the described program ImGQfinder were identified 3243 chetyrehrjadnye (n=4) ImGQ. Moreover, only 17% of them is perfect GQ (N=G) and can be identified using the previously known algorithms (Example 2. Figure 4A). Thus, the analysis of the sequence only one chromosome helped identify more than 2.5 thousand new potentially highly stable non-canonical structures. The analysis found sequences ImGQ showed that in nature replacement can meet both internal (ImEn, 48%)and external (ImEx, 35%) planes patterns. Moreover, the replacement G nucleotide N most often presents T and And and rare (less than 13%) (Figure 4A-C).

To confirm the formation of the non-canonical structures ImGQ and investigation of their thermal stability under physiological conditions were synthesized oligonucleotides bclGQ (sequence 3) and Ct1 (sequence 7). The sequence of oligomers apply to the natural sites found by the program ImGQfinder, the promoter region of the gene BCL-2 (one of the key genes of carcinogenesis) and intron a gene CTIF (component SWR/SUR complex of translation initiation [22]). The study results confirmed that the oligomers really form a highly stable (T melting point >70 C), steric-employed non-canonical structure ImGQ (Examples 1.1-1.5. Figures 2 and 3).

On the basis of the discovered rules of formation ImGQ were designed and received oligomers, simulating different position and the nature mononucleotide changes in the composition of artificial ImGQ (table 2). Studies have shown that in all cases there was observed a stable ImGQ (Example 1, table 2, Figure 2-3)that can serve as a reliable proof of the adequacy of the developed rules of forming ImGQ and programs ImGQfinder.

The way of search and prediction of formation of new non tertiary structures of polynucleotides, namely imperfect G-QUADRUPLEX, potentially stable in physiological conditions, will allow to find new molecular targets and biomarkers of pathological human States, to develop the new drugs directed action.

Description of drawings

On the Figure 1 shows the scheme of the structure chetyrehspalnyh intramolecular GQ with antiparallel (a and B)parallel () and mixed (G) mutual location GGGG fragments of the nucleotide sequence. When replacing one of guanine in one plane G 4 to another natural or synthetic nucleotide (examples substitution positions greyed out) structure correspond potentially stable ImGQ. L1, L2, L3 - fragments any sequence of nucleotide composition included loops GQ.

The Figure 2 shows the spectra KD GQ and ImGQ two natural fragments of the human genome - the promoter of the gene Bcl-2 (a) and intron a gene CTIF (D) - and their mutants (100 mM KCl, 10 mm TrisHCl, pH 7.5). The scheme of parallel QUADRUPLEX with two imperfect tetrad presented in Figure 2B. Specified external (endo-) and internal (Exo-) G 4 plane quadruplexes. Figure 2B shows the differential spectra melting GQ Ctg (sequence 2) and ImGQ Ct1 (sequence 7), Ct2, Ct3 and Ct4 (9-11 sequences), as in Figure 2 presents spectra KD GQ and ImGQ gene intron CTIF and his positional mutants obtained in the presence of 100 mM lithium ion, prevent the formation of GQ patterns. The Figure 2 shows the dependence of the rotational correlation intercalator (ethidium bromide) in complexes with GQ1 (sequence 8) (the control), bclGQ (sequence 3), TrGQ (sequence 6), Ct1 (sequence 7), Ct2 (sequence 9), Ct3 (sequence 10) and Ctg (sequence 2) of the volume of their molecules (the number of nucleotides).

The Figure 3 shows a comparison of H 1 NMR spectra of 2-Quartet QUADRUPLEX GQ1 (sequence 2) and oligomer Ct1 (sequence 7) - ImGQ. The rooms were eight proton signals of the two G4 planes in the spectrum GQ1 (sequence 2) (range at the bottom of the figure) and 14 of the signals of the four G4 planes ImGQ Ct1 (sequence 7) (range at the top of the figure), which in accordance with the previous approaches was also contain only 8 proton signals in the field of 12 ppm.

The Figure 4 shows the results of the analysis of the sequence of chromosome 18 person using a new algorithm of search sites, able to create new non-canonical structure - ImGQ. Presents a histogram of the distribution in the sequence 18 human chromosome sites that are able to form 4-Quartet committed GQ (Perfect GQ) and imperfect ImGQ carrying a single nucleotide substitution (A). Histogram B and reflect the composition mononucleotide replacement in sites ImGQ 18 chromosomes and their distribution in the external (ImEn) and internal (ImEx) planes structures.

Description of examples of implementation of the invention

Example 1 describes receive oligodeoxiribonucleotides natural and model sequences are shown in Table 2, (Example 1.1) and study their thermodynamic and spectral properties (Examples 1.2-1.4).

In the Examples 1.2-1.3 shows high stability of natural imperfect GQ from their promoter region of the gene BCL-2 and gene intron CTIF under physiological conditions. The evidence of the existence of the oligonucleotide sequence bcl2 ImGQ (sequence 1) in the solution in the form of a defective 4-Quartet, not perfect 3-Quartet GQ, was found in the standard algorithm [16] using the program quadfinder. Studied education, double-defective quadruplexes - derivatives ImGQ bcl2 (sequences 2-5) (table 2).

Similar results were obtained for artificial man-made sequences CC2, CC3, CC4 (sequences 9-11), Cta (sequence 12), Ctc (sequence 13) and shows that they form ImGQ and oligomer Ctg (sequence 2) - perfect GQ, which fully corresponds to our calculations.

Typical for GQ influence of potassium ions (stabilization) and lithium (destabilization) was observed for ImGQ. KD-spectra of oligonukleotidov Ct2, Ct3 and Ct4 (sequences 9-11) - positional isomers of natural Ct1 and perfect GQ Ctg obtained by replacing T in third position Ct1 on G spectra correspond GQ with various contributions parallel and antiparallel structures in the presence of K + (Figure 2D). The presence of Li + suppresses the production of non-canonical structures that clearly reflect similar spectra of oligomers in the Figure 2G.

Example 1.4. describes the experiments confirming the formation of intramolecular structures. Monomolekulyarnogo of quadruplexes follows from the data given on the Figure 2E, reflecting the proportional change of rotational relaxation complexes oligomers bromide by ethidium volume of the molecules, i.e. the number of nucleotide units in the composition of oligomers. In addition, the independence of the indicators of melting temperature ImGQ on their concentration also indicates the formation of molecular conformations.

Example 1.5. describes getting H 1 NMR spectra of 2-Quartet GQ 5'-GGGAGGCTGAGGCAGG (GQ1) and oligomer Ct1. On the basis of previously known representations of the oligomer Ct1 must also provide 2-Quartet GQ because the maximum length of four G-units in its structure does not exceed two. However, unlike the spectrum GQ1 presented in the field of 12 ppm 8 signals, corresponding to the two G 4-planes in the spectrum Ct1 observed 14 signal (region 12 ppm). This fact confirms the assumption that oligonucleotide Ct1 forms ImGQ-conformation consisting of three 4 G and one 3 G T (see diagram on Figure 1A, B).

Thus, education imperfect 4-Quartet ImGQ proven set of experimental data, including the formation of ImGQ follows from the significant differences of temperatures of melting and CD-spectra of oligonucleotides, the sequence of which correspond full and shortened bcl2 PQS (MP. bclGQ ~82°C >> MP. trGQ ~58°C, trGQ = tecwarranty GQ, a short-cut), thermodynamic and spectral characteristics (table 2, Figure 2A and Figure 3).

Table 2. The melting temperature bcl2 ImGQ and its derivatives in Tris-HCl buffer (pH 7.5).

Example 2 presents the results of the analysis of the primary sequence of the direct and inverse chains chromosome 18 person (total length is ~140 million nucleotides) ImGQ found using the algorithm and program ImGQfinder and their comparison with the results of the search in GQ environment known programs quadfinder.

Example 2.1 describes the search options

Example 2.2 describes the data processing and mapping of occurrence G/A, G/T and G/C replacement in imperfect QUADRUPLEX. Also in example 2.2 a comparison of the amount advanced and montevecchi imperfect 4-Quartet GQ 18 chromosome (Figure 4A-C).

It is shown that the most frequent replacement G/T (47% of the total number ImGQ). Replace G/A is also well represented (23% of the total number GQ). Replace the G/C presents a lesser extent (13%). Replace internal (Central) quartets prevail (48% of the total number of changes).

The share of committed GQ accounts for only 17% of the total number of potentially stable quadruplexes. Thus, the main part of the possible 4-Quartet non-canonical structures (more than three-quarters) was identified as a result of application of the new algorithm. The obtained data were the basis for the studies of the mechanisms of genome functioning and are an important new molecular targets for drug development directed action.

The following are examples of implementation of the invention.

Examples of implementation of the invention

Example 1. Getting oligodeoxiribonucleotides natural and model sequences (Table 2) and study their thermodynamic and spectral properties.

Example 1.1. Synthesis of oligodeoxyribonucleotides

Solid-phase method was used on automatic DNA synthesizer AFM 800 ("Bisset", Russia) were obtained oligonucleotides, the sequence of which is given in Table 2.

Example 1.2. Determination of thermodynamic parameters of education conformers DNA profiles of thermal dissociation. Spectra circular dichroism of oligonucleotides.

Profiles of thermal dissociation of quadruplexes were registered on spectrometer Jasco V-550 (USA), equipped with thermostatic console, with λ=295 nm in the temperature range from 20 to 90 C. the Melting at a given wavelength considered proof of quadruplexes patterns. Thermodynamic parameters of education quadruplexes, in particular melting point was calculated by the method of nonlinear regression in the program KaleidaGraph v. 4.0 (Synergy, UK), based on the model of two States (1).

CD spectra of oligonucleotides were registered on spectropolarimetry Jasco 715 (USA) with thermostatted cell at the temperature of 20 C in the range of wavelengths 220-330 nm.

Immediately before registration KD spectra and melting curves solutions oligonucleotides 20 mm Tris-HCl buffer (pH 7.5) with different concentrations of KCl and LiCl was heated to 90 degrees C, kept at this temperature for 3 minutes and quickly cooled to 0 C.

Example 1.3. The composition of the stable steric conformations of oligonucleotides potential ImGQ on rotational relaxation complexes of the ethidium bromide (EtBr) : QUADRUPLEX.

Rotational relaxation (p) complexes was calculated by the equation Perrin-Weber [23]:

where P is observed fluorescence polarization EtBr in complex with oligonucleotide, d =41 of + / -1% - polarization in the absence of rotational diffusion, t - lifetime fluorescence EtBr in complex with oligonucleotide.

The fluorescence polarization EtBr in complex with oligonucleotide was determined by the ratio of the intensities of vertical and horizontal component of fluorescence at excitation vertically polarised light. The intensity of fluorescence (?=610 nm) was measured at an Cary Eclipse (Varian, USA) when excited by light with a wavelength of 540 nm at 20 C. The fluorescence polarization was calculated by the formula [24]:

P=(I ║ -I ┴ )/(I ║ +I ┴ )

The lifetime fluorescence complexes EtBr : QUADRUPLEX was determined with the help of an Easy Life V (Optical Building Blocks, USA) in pulse mode.

Example 1.4. H 1 NMR spectra of oligonucleotides received (0.1 mm solution of the oligonucleotide in water, H 2 O/D 2 O, 9/1, 100 mM KCl, 20 mM TrisHCl, pH 7.5). The spectra were obtained using the device Bruker AMX400 (Germany), at the temperature of 19 degrees C with signal suppression water. For the interpretation of the spectra used the program MestReNova version 7.0.3 (Mestrelab Research SL, Spain).

Example 2. The description of the algorithm of search ImGQ and its application for the analysis of primary nucleotide sequences and design of artificial DNA fragments potential ImGQ.

Example 2.1. The description of the algorithm of search sites potential ImGQ as part of polynucleotides.

Formula sequences of fragments of polynucleotides (ImGQ), can potentially take a conformation imperfect quadruplexes shown in Table 1.

In the preparation of the algorithm takes into account only intramolecular ImGQ that can exist simultaneously. For example, a fragment GGGTGG is recorded only once, excluding the possibility of implementing alternative conformations.

For a sample stable in physiological terms of sequences imperfect GQ were introduced the following boundary conditions:

- to consider only mono-imperfect ImGQ, i.e. in all planes GQ only one possible replacement of guanine to another nucleotide;

- n > 4 (n - the number of quartets formed four consecutive G-rich snippets sequence);

- x=1 to 7 (x - length of loops as part ImGQ);

For N=G, the formula describes a perfect GQ.

On the basis of this algorithm written program search stable ImGQ in primary nucleotide sequences - ImGQfinder (Application for State registration from 06.09.2012)

Example 2.2. Search GQ and potentially stable ImGQ in the primary sequence of chromosome 18 were performed using ImGQFinder (Application for State registration of computer programs from 06.09.2012).

Analyzed the sequence of chromosome 18 man from NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/NC_000018.9) Search ImGQ conducted separately for direct and inverse circuits. Considered perfect and montevecchia 4-Quartet GQ. Each multivariate GQ (PQS that can form several GQ different structure) was taken into account only once. This was achieved by mapping coordinates found GQ. When crossing of coordinates defective and committed GQ defective QUADRUPLEX cast, fixed perfect as thermodynamically more likely.

Comparing the representation of perfect and imperfect GQ 18 chromosome. Determination of the frequency of occurrence of different types of defects in imperfect GQ.

Counted separately the amount committed and GQ GQ with substitutions G/A, G/C and G/T. the search Results on direct and inverse circuits summarized. The number of GQ with external defects was calculated as the sum of substitutions in the first and fourth quartets, with internal defects - as a sum of substitutions in the second and third quartets.

Literature

1. Raiber E.A., Kranaster R., Lam E., Nikan M. S. Balasubramanian A non-canonical DNA structure is a binding motif for the reduced factor SP1 in vitro // Nucleic Acids Res. - 2011.

2. Kouzine F. Levens D. Supercoil-driven DNA structures regulate genetic transactions // Front Biosci. - 2007; 12 4409-4423.

3. Brown R.V. Hurley L.H. DNA acting like RNA // Biochem Soc Trans. - 2011; 39 (2): 635-640.

4. Nelson L.D., C. Bender, Mannsperger H., Buergy D., Kambakamba P., Mudduluru G., Korf u., D. Hughes, M.W. Van Dyke Allgayer H. Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer // Mol Cancer. - 2012; 11 (1): 38.

5. Cer THIS, Bruce K.H., Donohue D.E., Temiz N.A., Mudunuri U.S., Yi M., Volfovsky N., Bacolla A., Luke B.T., Collins J.R. Stephens R. Searching for non-B DNA-forming motifs using nBMST (non-B DNA motif search tool) // Curr Protoc Hum Genet. - 2012; Chapter 18 Unit 18 17 11-22.

6. Shchyolkina A.K., Borisova O.F., Livshits M.A., Pozmogova G.E., Chernov B.K., Klement R. Jovin T.M. Parallel-stranded DNA with mixed AT/GC composition: the role of trans G.C base pairs in sequence dependent helical stability // Biochemistry. - 2000; 39 (33): 10034-10044.

7. Hsu S.T., Varnai P., Bugaut A., Reszka A.P., Neidle S. S. Balasubramanian A G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex having asymmetric G-tetrad dynamics // J Am Chem Soc. - 2009; 131 (37): 13399-13409.

8. Shahid R., Bugaut A. S. Balasubramanian The BCL-2 5' untranslated region contains an RNA G-quadruplex-forming motif that modulates protein expression // Biochemistry. - 2011; 49 (38): 8300-8306.

9. Tolmachov O.E. Self-entangled of long linear DNA vectors using transient non-B-DNA attachment points: A new concept for improvement of non-viral therapeutic gene delivery // Med Hypotheses. - 2012.

10. Fisette J.F., Montagna D.R., Mihailescu M.R. Wolfe M.S. A G-Rich element forms a G-quadruplex and regulates BACE1 mRNA alternative splicing // J Neurochem. - 2012.

11. Cheng L.C., Pai T.W. Li L.A. Regulation of human CYP11B1 and CYP11B2 promoters by transposable elements and conserved cis elements // Steroids. - 2012; 77 (1-2): 100-109.

12. W. Zhou, Brand N.J. Ying L. G-quadruplexes-novel preparation of gene function // J Cardiovasc Transl Res. - 2011; 4 (3): 256-270.

13. Huppert J.L. S. Balasubramanian G-quadruplexes in promoters throughout the human genome // Nucleic Acids Res. - 2007; 35 (2): 406-413.

14. Wu Y. Brosh R., Jr. G-quadruplex nucleic acids and human disease // FEBS j - 2010; 277 (17): 3470-3488.

15. S. Balasubramanian, Hurley L.H. Neidle S. Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? // Nat Rev Drug Discov. - 2011; 10 (4): 261-275.

16. Huppert J.L. S. Balasubramanian Prevalence of quadruplexes in the human genome // Nucleic Acids Res. - 2005; 33 (9): 2908-2916.

17. Gonzalez V., K. Guo, Hurley L. Sun D. Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein // J Biol Chem. - 2009; 284 (35): 23622-23635.

18. Dai J., Dexheimer T.S., D. Chen, M. Carver, Ambrus A., R.A. Jones Yang D. An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution // J Am Chem Soc. - 2006; 128 (4): 1096-1098.

19. Zaitseva M, Kaluzhny D., Shchyolkina A., O. Borisova, I. Smirnov Pozmogova G. Conformation and thermostability of oligonucleotide d(GGTTGGTGTGGTTGG) containing thiophosphoryl internucleotide bonds at different positions // Biophysical Chemistry. - 2010; 146 (1): 1-6.

20. Hazel P., Huppert J., S. Balasubramanian Neidle S. Loop-length-dependent folding of G-quadruplexes // J Am Chem Soc. - 2004; 126 (50): 16405-16415.

21. Guedin A., De Cian A., Gros j, Lacroix L. Mergny J.L. Sequence effects in single-base loops for quadruplexes // Biochimie. - 2008; 90 (5): 686-696.

22. Kim C.M., H. Cho, K. Choi, J. Kim, B.W. Kim, To Y.G., Jang S.K. Kim Y.K. A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation // Genes Dev. - 2009; 23 (17): 2033-2045.

23. Marky L.A. Breslauer K.J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves // Biopolymers. - 1987; 26 (9): 1601-1620.

24. Weber G. Anderson S.R. The effects of energy transfer and rotational diffusion upon the fluorescence polarization of macromolecules // Biochemistry. - 1969; 8 (1): 361-371.

The method of obtaining for man-made synthetic oligonucleotides, potentially able to form stable in physiological and close to physiological conditions of the non-canonical structure is imperfect G-QUADRUPLEX (ImGQ), including single nucleotide substitution in G 4 planes in the G-QUADRUPLEX (GQ), including the use of the algorithm descriptions of the nucleotide sequences in the form of a set of the following structural formula

(for k = 4; x=1 to 7), where k is the number of the Quartet formed four consecutive G - rich snippets nucleotide sequence; L - any nucleotide in the loop ImGQ; x is the number of nucleotides in the loop; N is any not-G nucleotide in the plane ImGQ; for further synthesis of man-made synthetic oligonucleotides, potentially capable of forming ImGQ and characterized by the selected sequences.