DE19842527A1 - Formation of a triple helix forming oligomer comprises searching for a cytosine and/or adenine-rich region of a double-stranded DNA - Google Patents

Abstract

Formation of a triple helix forming oligomer by searching for a cytosine and/or adenine-rich region of a double-stranded DNA is new. A method for formation of a triple helix forming oligomer and its compounds comprises searching for a region with a suitable sequence from a nucleic acid strand, preferably a DNA double strand, derived from the oligomer sequence. The region is chosen from a cytosine and/or adenine rich sequence and the oligomer, particularly a compound, is synthesized, by agreement of the oligomer sequence with the sequence of the chosen region of the nucleic acid strand, inclusive of the strand orientation.

Description

The invention relates to a method for forming triple helix-forming Oligomers and their compounds, in the section provided a region with a suitable sequence of a DNA duplex from which the oligomer sequence is derived.

Triple helix-forming oligonucleotides include the following Areas of application found: Mega base cutting (Strobel S. A., Dervan P. B., Methods Enzymol 1992, 216, 309-321; Strobel S.A., Doucette strain L.A., Riba L., Housman D.E., Dervan P.B., Science 1991, 254, 1639-1642; Strobel S.A., Dervan P.B., Nature 1991, 350, 172-174; Strobel S.A., Dervan P. B., Science 1990, 249, 73-75), transcription inhibition (Hiratou T., Tsukahara S., Takai K., Koyanagi Y., Yamamoto N., Takaku H., Nucleic Acids Symp Ser 1997, 37, 221-222; Kim H. G, Reddoch J. F., Mayfield C., Ebbinghaus S., Vigneswaran N., Thomas S., Jones D. E. Jr, Miller D. M., Biochemistry 1998, 37, 2299-2304; Curcio L.D., Bouffard D.Y., Scanlon K. J., Pharmacol Ther 1997, 74, 317-332; Grigoriev M., Praseuth D., Robin P., Hemar A., Saison-Behmoaras T., Dautry-Varsat A., Thuong N. T., Helene C., Harel-Bellan A., J Biol Chem 1992, 267, 3389-3395; Duval-Valentin G., Thuong N.T., Helene C., Proc Natl Acad Sci USA 1992, 89, 504-508; Helene C., Anticancer Drug Des 1991, 6, 569-584), mutagenicity generation and -Proof (Bacolla A., Ulrich M. J., Larson J. E., Ley T. J., Wells R. D., J Biol Chem 1995, 270, 24556-24563; Olivas W. M., Maher L. J., Biotechniques 1994, 16, 128-132; Ulrich M. J., Gray W. J., Ley T. J., J Biol  Chem 1992, 267, 18649-18658), separation process or cleaning (Seeger C., Batz H.G., Orum H., Biotechniques 1997, 23, 512-517; Kiyama R., Nishikawa N., Oishi M., JMoI Biol 1994, 237, 193-200).

In total, the following types of nucleic acid triple helices are so far have been described.

1. Pyrimidine motif

This motif is characterized by the following structure: The double strand consists in the triple helical part essentially of a homopurin strand and from a homopyrimidine strand. The third strand is in the major groove of the double strand and is a homopyrimidine strand. Whose The sequence is analogous, but the orientation is reversed from that of the Double stranded homopyrimidine (Bartley J.P., Brown T., Lane A.N., Biochemistry 1997, 36, 14502-14511; Koshlap K.M., Schultze P., Brunar H., Dervan P.B., Feigon J., Biochemistry 1997, 36, 2659-2668; Wang E., Koshlap K.M., Gillespie P., Dervan P.B., Feigon J., J Mol Biol 1996, 257, 1052-1069; Radhakrishnan L, Patel D.J., J Mol Biol 1994, 241, 600-619; Radhakrishnan L, Patel D.J., Structure 1994, 2, 395-405; Radhakrishnan L, Patel D.J., Structure 1994, 2, 17-32; Radhakrishnan L, Patel D.J., Priestly E. S., Nash H. M., Dervan P. B., Biochemistry 1993, 32, 11228-11234).

2. Purine motif

This motif is characterized by the following structure: The double strand consists in the triple helical part essentially of a homopurin strand and from a homopyrimidine strand. The third strand is in the major furrow of the double strand and is a homopurine strand. Whose The sequence is analogous, but the orientation is reversed from that of the Homopurin strands in a double strand (Ji J., Hogan M. E., Gao X., Structure 1996, 4, 425-435, Radhakrishnan L, Patel D.J., Structure 1994, 2, 395-405;  

Dittrich K., Gu J., Tinder R., Hogan M., Gao X., Biochemistry 1994, 33, 4111-4120; Radhakrishnan L, Patel D.J., Strueture 1993, 1, 135-152).

3. Mixed form

This motif is characterized by the following structure: The double strand consists of a strand rich in purines and in the triple helical part a complementary strand. The third strand is in the big one Furrow of the double strand and is made up of guanines and thymines (Radhakrishnan L, Patel D.J., Structure 1993, 1, 135-152). Both orias The third strand can be compared to the double strand. In the extended mixed form, which also contains adenine, the Orientation antiparallel to the duplex homopurin strand.

The types 1 to 3 have in common that the bases of the third strand in essential only with the bases of the purine-rich strand in the double strand in Interaction.

4. R-shape or recombination motif

This motif is characterized by the following structure: The double strand is not subject to any sequence restriction in the triple helical part. The third Strand is in the large furrow of the double strand. It is like one of the strands of the double strand in the orientation and largely in the sequence. The bases of the third strand occur with the base pairs of the double strand interacting (Kiran M. R., Bansal M., J Biomol Struct Dyn 1997, 15, 333-345; Kim M.G., Zhurkin V.B., Jernigan R.L., Camerini-Otero R.D., J Mol Biol 1995, 247, 874-889; Zhurkin V.B., Raghunathan G., Ulyanov N.B., Camerini-Otero R.D., Jernigan R.L. J Mol Biol 1994, 239, 181-200).

The spontaneous formation of this motif from single strands without the participation of Proteins (recombinases) or peptides (partial sequences from the Recombinases, Voloshin O., Wang L., Camerini-Otero R. D .; Promotion of  homologous DNA pairing by RecA-derived peptides, US 5,731,411) has not been described so far.

For the creation of a suitable sequence of oligonucleotides that spontaneously form triple-helical structures with double-stranded DNA, the longest possible and undisturbed homopurin-homopyrimidine sequence is sought in the target area of the DNA:

• a) An oligonucleotide is synthesized which contains the same pyrimidines in the same sequence as the homopyrimidine strand but in reverse Includes strand orientation. Cytosines may be exchanged against analogues (5-methylcytosine, 8-oxoadenine, pseudoisocytosine, 2-amino pyridine, 5-propynylcytosine, etc.). Where the homopyrimidine is interrupted by an adenine, a guanine is inserted into the Oligonucleotide set; where the homopyrimidine sequence is through a Guanine is interrupted, a thymine or a cytosine is added to it Oligonucleotide set or
• b) an oligonucleotide is synthesized which contains the same purines in the same sequence as the homopurin strand but in reverse Includes strand orientation. At the places where the homopurin sequence goes through if a cytosine is interrupted, a thymine is set; where the Homopurin sequence is interrupted by a thymine, a cytosine is in set the oligonucleotide. Possibly some or all Adenine replaced by thymine.

J.R. Fresco (US 5,693,471 Triple stranded Nucleic acids) claims one Extension of the procedure under b) by adding a purine sequence in the first strand an occurring adenine not just an adenine or an Thymine, but also a uracil or a hypoxanthine, not a guanine  just a guanine, but also a cytosine or hypoxanthine so that the third strand is in the area where the first one has pure purine sequence itself, neither a pure homopurin sequence nor a pure one Homopyrimidine sequence.

Disadvantages of the previously known motifs:

• 1. The triplex types 1.-3. are on duplex sequences of homopurin / Homopyrimidine limited. This makes all the gene sequences of one Influenced by triple helix-forming oligonucleotides excluded such a sequence neither in the coding nor in the regulatory area have sufficient length.
• 2. Under physiological conditions, types 1.-4. the stability in usually significantly lower than the corresponding duplex stability. This means high use concentrations of triple helix-forming oligonucleo tide.
• 3. Any violation of the duplex homosequence results in triple helices of the types 1.-3., Which have a lower stability. That also means that it does corresponding triple helix-forming oligonucleotide more binding There are duplex sequences (ambiguity in the target sequence).
• 4. Type 1 triple helices usually require a pH of 6 or below that, they do not exist under physiological conditions. The This is due to the cytosines, which are only protonated at N3 corresponding hydrogen bonds with the guanine in the duplex can train. Due to the charge of protonated cytosines Sequences with neighboring cytosines are less suitable.
• 5. Type 4 triple helices have such a low stability that they cannot form spontaneously. So far they have only been available with the help of proteins (e.g. RecA from E. coli) or intramolecularly, that is, by linking the third strand was generated by means of a linker with the duplex.  
• 6. Triple helix-forming oligonucleotides of type two with very high percentage of purines and tend to compete complexes. They form duplexes parallel to each other or with a high proportion of Guanine also tetraplexes (guanine tetrades).

Cohen J. S. and Hogan M. E. (Spectrum of Science, 1995, 2, 28-34) estimate that only about every second section of DNA is for triple helix contains necessary sequence sections.

The object of the invention is a process for the formation of such oligomers and to create their connections, the triple helices of high stability and Selectivity with double-stranded DNA form under physiological Conditions and in areas where the duplex sequences are also different than the homopurine-homopyrimidine structure.

According to the invention, triple helix-forming oligomers and their Connections created by a region of a from the DNA double strand Nucleic acid strand is selected that is rich in cytosines and / or rich is on adenines, and there are oligomers or their compound synthesized in which the oligomer sequence matches the sequence of the selected area of the nucleic acid strand, including the strand orientation, agrees. Thus, triple helix-forming oligomers and their connections also created for such nucleic acid regions in which neither a homopurine nor a homopyrimidine sequence is present. The result is oligomers and their compounds that are rich on cytosine or its analogs and / or on adenine or its analogs are with a hairpin duplex, one strand in sequence and 5'-3 'orientation matches the oligomer, three-stranded structures,  what remarkable properties regarding structural stability and May have sequence variability.

Advantageous method configurations are in the subclaims 2 to 11 called.

The invention is based on exemplary embodiments and Illustrations are explained in more detail.

Show it:

Fig. 1: Thermal melting curves in sum and components illustration of a Einzelstrangoligonukleotids 5'-d (A ₁₂₎ -3 '(SEQ ID NO: 1) and a Haarnadeloligonukleotids (double strand) 5'-d (A ₄ C ₁₂ T ₁₂ ) -3 '(SEQ ID-NO: 9)

FIG. 2: Job plot at T = 30 ° C. of the system according to FIG. 1

FIG. 3 shows concentration dependence of the melting temperature Tm for system of Figure 1 and a simple sequence extensions.

Fig. 4: melting temperatures of the triple helical complex of single and double strand at 2 micromolar complex concentration depending on base pair (in the double strand) and base exchange (in the single strand) at suitable positions

Legends for the figures

Fig. 1:
Solid line: The absorbance at 260 nm is plotted as a function of the Celsius temperature for an equimolar solution of a single-stranded oligonucleotide 5'-d (A ₁₂ ) -3 '(SEQ ID-NO: 1) and a hairpin-oligonucleotide (double-stranded) S'-d (A ₁₂ C ₄ T ₁₂ ) -3 '(SEQ ID-NO: 9), in which the two complementary strands are connected by four cytosines, in 10 mmol / l sodium cacodylate, 200 mmol / l sodium chloride and 0.1 mmol / l EDTA. The concentration of each oligonucleotide is 3 µmol / l.
Dotted line: Melting curve of the single strand 5'-d (A ₁₂ ) -3 '(SEQ ID NO: 1) in the same medium and in the same concentration, but shifted so that it coincides with the solid melting curve at 11 ° C.
Narrow dashed line: melting curve of the hairpin double strand 5'-d (A ₁₂ C ₄ T ₁₂ ) -3 '(SEQ ID-NO: 9) in the same medium and in the same concentration, but shifted so that it is at 11 ° C matches the solid melting curve.
Broken line: sum of the melting curves of single strand 5'-d (A ₁₂ ) -3 '(SEQ ID-NO: 1) and hairpin double strand 5'-d (A ₁₂ C ₄ T ₁₂ ) -3' (SEQ ID -NO: 9) in the same medium and in the same concentration, but shifted so that it coincides with the solid melting curve at 40 ° C.
The shift is only 1.4% of the final level.

Fig. 2:
Job plot of the system 5'-d (A ₁₂ ) -3 '(SEQ ID-NO: 1) and 5'-d (A ₁₂ C ₄ T ₁₂ ) -3' (SEQ ID-NO: 9) at 30 ° C, milieu conditions as in Fig. 1. The absorbance measurements were carried out twice or three times. The sum of the two oligonucleotide concentrations was recorded at 4 µmol / l. The differences between the averaged measured values are plotted against a linear function (1-x) E (A ₁₂ ) + x E (A ₁₂ C ₄ T ₁₂ ), E (A ₁₂ ) and E (A ₁₂ C ₄ T ₁₂ ) being the Absorbances of the four micromolar solutions of the individual oligonucleotides are and x varies between 0 and 1.
Checked, dashed line: measurements at 280 nm,
Circle, solid line: measurements at 220 nm,
Triangle, dotted line: measurements at 260 nm.
The lines correspond to the best adjustments of a simply bent straight line to the measured values, full symbols have not been taken into account in the adjustment.

Fig. 3:
a: First derivations of the smoothed melting curves of the system 5'-d (A ₁₂ ) -3 '(SEQ ID-NO: 1) and 5'-d (A ₁₂ C ₄ T ₁₂ ) -3' (SEQ ID-NO: 9), each diluted by a factor of 2 in four steps, multiplied by the dilution factor (n = 1, 2, 4, 8, 16).
b: Triplex melting temperatures of four different double-strand single-strand combinations from dilution series as a function of the concentration. Continuous lines: Best adjustments of the measured values by the formula Tm = ΔH / (ΔS + R.ln (c / 2)) - 273.16 (Tm is given in degrees Celsius, the general gas constant R in the same units as ΔS).
dashed lines: associated trust hose (+ - standard deviations).
Full circles: 5'-d (A ₁₂ C ₄ T ₁₂ ) -3 '+ 5'-d (A ₁₂ ) -3', (SEQ ID-NO: 9, 1)
Squares: 5'-d (A ₄ TA7C ₄ T7AT ₄ ) -3 '+ 5'-d (A ₄ TA7) -3', (SEQ ID-NO: 12, 5)
Triangles: 5'-d (A ₄ GA7C ₄ T7CT ₄ ) -3 '+ 5'-d (A ₄ GA7) -3', (SEQ ID-NO: 10, 3)
Checks: 5'-d (A ₄ CA7C ₄ T7GT ₄ ) -3 '+ 5'-d (A ₄ CA7) -3', (SEQ ID-NO: 11, 4)
additionally
open circles: 5'-d (T ₁₂ ) -3 '+ 5'-d (A ₁₂ ) -3' (SEQ ID-NO: 8, 1).

Fig. 4:
a: System hairpin double strand with position XY, which can be occupied by any Watson-Crick base pair and single strand with position Z, which can be replaced by any of the four common bases.
b: Columns refer to the Watson-Crick base pair below (XY), the symbols refer to the base in the single strand (Z). The Tm values for the respective combinations base pair (XY, column) and base (Z, symbol) can be read off the ordinate.

Embodiments

In the following examples, interpolated values for melting temperatures, which correspond to an oligonucleotide concentration of two micromolar each, are indicated with errors. These simple errors correspond to the standard deviations of the values calculated for this concentration from the adjustments of the observed dependencies of the Tm values on the concentrations according to the formula given in the legend to FIG. 3. The number of degrees of freedom is usually 2 to 3. For a statistically justified error, the corresponding fractiles of the student distribution would have to be taken into account. Example 12 has been implemented in two independent implementations. The difference between the Tm values from 0.5 to 0.6 K found there can be assumed to be a realistic error.

example 1

A single strand oligonucleotide 5'-d (A ₄ GA ₇ ) -3 '(SEQ ID-NO: 3) and a hairpin duplex 5'-d (A ₄ GA ₇ C ₄ T ₇ CT ₄ ) -3' (SEQ ID-NO : 10) are dissolved equimolar in a buffer containing 10 mmol / l sodium cacodylate and 200 mmol / l sodium chloride and 0.1 mmol / l EDTA. The absorption of this solution at 260 nm is registered as a function of temperature. The resulting curve is smoothed numerically and derived according to the temperature. The first maximum of this first derivative is interpreted as the melting temperature Tm of the complex consisting of single strand and duplex hairpin. In a 1: 1 dilution series, the equimolar concentrations vary between 3.1 and 0.19 micromolar. A Tm value of 30.22 + -0.02 ° C is determined for a two-micro molar solution.

Example 2

As example 1, only with the following differences:

The single strand is 5'-d (A ₇ GA ₄ ) -3 '(SEQ ID-NO: 2). The equimolar solutions with the same hairpin duplex vary in concentration between 3.15 and 0.20 micromolar. A Tm value of 20.11 + -0.05 ° C is determined for a two micromolar solution.

Example 3

As example 1, only with the following differences:

The single strand is 5'-d (A ₁₂ ) -3 '(SEQ ID-NO: 1), the hairpin duplex 5'-d (A ₁₂ C ₄ T ₁₂ ) -3' (SEQ ID-NO: 9). The equimolar solutions vary in concentration between 3.0 and 0.19 micromolar. A Tm of 31.78 + -0.24 ° C is determined for a two micromolar solution.

Example 4

As example 1, only with the following differences:

The single strand is 5'-d (A ₄ CA ₇ ) -3 '(SEQ ID-NO: 4), the hairpin duplex 5'- d (A ₄ CA ₇ C ₄ T ₇ GT ₄ ) -3' (SEQ ID- NO: 11). The equimolar solutions vary in concentration between 3.0 and 0.19 micromolar. A Tm value of 33.46 + -0.07 ° C is determined for a two-micro molar solution.

Example 5

As example 1, only with the following differences:

The single strand is 5'-d (A ₄ TA ₇ ) -3 '(SEQ ID-NO: 5), the hairpin duplex 5'- d (A ₄ TA ₇ C ₄ T ₇ AT ₄ ) -3' (SEQ ID- NO: 12). The equimolar solutions vary in concentration between 3.5 and 0.22 micromolar. A Tm value of 28.78 + -0.20 ° C is determined for a two-micro molar solution.

Example 6

As example 5, only with the following differences:

The hairpin duplex is 5'-d (A ₄ CA ₇ C ₄ T ₇ GT ₄ ) -3 '(SEQ ID-N0: 11). The equimolar solutions with the same single strand vary in concentration between 3.0 and 0.19 micromolar. A Tm of 21.92 + -0.36 ° C is determined for a two micromolar solution.

Example 7

As example 5, only with the following differences:

The hairpin duplex is 5'-d (A ₄ GA ₇ C ₄ T ₇ CT ₄ ) -3 '(SEQ ID-NO: 10). The equimolar solutions with the same single strand vary in concentration between 3.0 and 0.19 micromolar. A Tm of 16.98 + -0.61 ° C is determined for a two micromolar solution.

Example 8

As example 4, only with the following differences:

The hairpin duplex is 5'-d (A ₁₂ C ₄ T ₁₂ ) -3 '(SEQ ID-NO: 9). The equimolar solutions with the same single strand vary in concentration between 3.3 and 0.21 micromolar. A Tm of 18.47 + -0.46 ° C is determined for a two micromolar solution.

Example 9

As example 3, only with the following differences:

The hairpin duplex is 5'-d (A ₄ TA ₇ C ₄ T ₇ A ₄ ) -3 '(SEQ ID-NO: 12). The equimolar solutions with the same single strand vary in concentration between 3.3 and 0.21 micromolar. A Tm value of 16.85 + -0.06 ° C is determined for a two micromolar solution.

Example 10

As example 1, only with the following differences:

The hairpin duplex is 5'-d (A ₄ TA ₇ C ₄ T ₇ A ₄ ) -3 '(SEQ ID-NO: 12). The equimolar solutions with the same single strand vary in concentration between 3.87 and 0.24 micromolar. A Tm value of 18.48 + -0.25 ° C is determined for a two-micro molar solution.

Example 11

As example 1, only with the following differences:

The hairpin duplex is 5'-d (A ₄ CA ₇ C ₄ G ₇ A ₄ ) -3 '(SEQ ID-NO: 11). The equimolar solutions with the same single strand vary in concentration between 3.2 and 0.2 micromolar. A Tm of 19.11 + -0.11 ° C is determined for a two micromolar solution.

Example 12

As example 1, only with the following differences:

The single strand is 5'-d ([CA] ₅ A2) -3 '(SEQ ID-NO: 6), the hairpin duplex 5'-d ([CA] ₅ A2C ₄ T2 [TG] ₅ ) -3' (SEQ ID-NO: 13). The equimolar solutions vary in concentration between 3.55 and 0.22 micromolar. A Tm value of 49.46 + -0.16 ° C is determined for a two micromolar solution. In an independent repeat measurement with a new sample, a Tm value of 50.02 + -0.29 ° C was determined.

Example 13

As example 1, only with the following differences:

The single strand is 5'-d (C ₅ A ₇ ) -3 '(SEQ ID-NO: 7), the hairpin duplex 5'-d (C ₅ A ₇ C ₄ T ₇ G ₅ ) -3' (SEQ ID- NO: 14). The equimolar solutions vary in concentration between 2.93 and 0.37 micromolar. A Tm value of 45.50 + -0.33 ° C is determined for a two micromolar solution.

SEQUENCE LOG

Claims (11)

1. A process for the formation of triple helix-forming oligomers and their compounds, in which a region with a suitable sequence is sought from a nucleic acid strand, preferably a DNA duplex, from which the oligomer sequence is derived, characterized in that a region of the nucleic acid strand is selected which is rich in cytosines and / or rich in adenines and that an oligomer or its compound is synthesized in which the oligomer sequence matches the sequence of the selected region of the nucleic acid strand, including the strand orientation. 2. The method according to claim 1, characterized in that an area of Nucleic acid strand is selected in which of the bases at least 17% cytosine, at least 17% adenine and in total at least 67% are adenines or cytosines. 3. The method according to claim 1, characterized in that an oligomer or whose compound is synthesized, in which the base completely or partially replaced by base analogs. 4. The method according to claim 3, characterized in that an oligomer or whose compound is synthesized, in which the base Cytosine is replaced by cytosine analogues, whereby to the cytosine analogues at least pseudoisocytosine, 5-methylcytosine, 5-propynylcytosine, 6-oxocyto sin, 2-aminopyridine, 8-oxoadenine and all six-ring systems are to be counted, which are exocyclic in relation to the ring position connected to the backbone Carry an oxygen in the 2-position or an amino group in the 4-position.   5. The method according to claim 3, characterized in that an oligomer or whose compound is synthesized, in which the base adenine is replaced by adenine analogs, at least to the adenine analogs Count 2-aminopurine, 2,6-diaminopurine and all compounds are on a Puring round body except substitutions on others Positions still in the 2- or 6-position exocyclic an amino group wear. 6. The method according to claim 3, characterized in that an oligomer or whose compound is synthesized, in which the base Thymine is replaced by thymine analogues, being among the thymine analogues to understand at least 5-propynyluracil, uracil and those compounds are in which one or both of the natural bases are thymine or Uracil are replaced by sulfur. 7. The method according to claim 3, characterized in that an oligomer or whose compound is synthesized, in which the base is guanine is replaced by guanine analogs, at least among guanine analogs 6-thioguanine is to be understood. 8. The method according to claim 1, characterized in that an oligomer lengths 7 to 40 are synthesized. 9. The method according to claim 1, characterized in that a Oligomer compound is synthesized, the oligomer portion of a length 5 owns up to 40.   10. The method according to claims 1 and 3, characterized in that the Oligomer or its compound in a predominantly unbranched arrangement Nucleic acid bases and / or base analogs with different backbones connects, including DNA, RNA, in the 2'-position or on the phosphorus modified backbones, peptide backbones (PNA) and backbones with Morpholino or guanidino structure. 11. The method according to claims 1 and 3, characterized in that a Oligomer compound is synthesized, which is preferably the compound of an oligomer with one or more chemical substances that show particular affinity for nucleic acid structures, and / or the Represents connection between oligomers.