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Justia Patents Trivalent Phosphorus Compound UtilizedUS Patent for Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed Patent (Patent # 10,844,087)
Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed
Oct 30, 2014 - JAPAN SCIENCE AND TECHNOLOGY AGENCY
Provided is a means for preventing the inactivation of a photoresponsive nucleic acid probe by suppressing the formation of a photocrosslink between a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III) and a modified nucleotide having a structure corresponding to the monomer of Formula (I), wherein the modified nucleotide replaces at least one constituent nucleotide which is the photocrosslinkable 1-thyminyl or 1-uracilyl, by substituting at least one constituent nucleotide which is the photocrosslinkable 1-thyminyl or 1-uracilyl with a modified nucleotide having a structure corresponding to the monomer of Formula (I).
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This application is a national stage of international application no. PCT/JP2014/078980, filed Oct. 30, 2014, and claims priority to Japanese application no. 2013-225799, filed Oct. 30, 2013, the entirety of all of which are herein incorporated by reference.
The present invention relates to a method of suppressing photo-crosslinkage of a photoresponsive base having a photo-crosslinkable vinyl structure with a photo-crosslinkable thymine (T) or uracil (U) base, and also relates to a photoresponsive nucleic acid with suppressed self-crosslinking ability.
As a basic technique in the field of molecular biology, formation and detection of a double strand of nucleic acids are widely used not only in basic research but also in the fields of medical care, industry, agriculture, and the like. A particularly useful technique used in formation and detection of a double-stranded nucleic acid is the photo-crosslinking technique with the use of photoresponsive nucleic acids. The photo-crosslinking technique is used in a wide range of applications, for example, in the field of medical care including antisense drugs and other nucleic acid drugs as well as SNP sensing, and the field of DNA nanotechnology with the use of nucleic acids. The photo-crosslinking technique with the use of photoresponsive nucleic acids has been developed by the inventors of the present invention and their research group, and a plurality of artificial photoresponsive nucleotides have been developed and are under patent pending (Patent Document 1).
One of the most remarkable applications of the photo-crosslinking technique is highly-sensitive selective amplification of a nucleic acid (the photo-clamping method) (Patent Document 2). This is a method of, prior to PCR amplification of a nucleic acid, using a photoresponsive nucleic acid as a clamp probe and subjecting the clamp probe to photo-linkage with a nucleic acid that has a wild-type (or normal) base sequence and is present in a large number in a specimen, so as to form an indissociable double-stranded nucleic acid, and as a result, suppressing PCR amplification of the large number of nucleic acid having a wild-type (or normal) base sequence, thereby allowing selective and highly sensitive amplification of a nucleic acid that has a target mutant base sequence and is present in a small amount in the specimen.
Patent Literature 1: International Publication No. WO 2009/066447
SUMMARY Technical Problem
The inventors of the present invention and their research group encountered a problem related to the photo-clamping method: a photoresponsive nucleic acid sometimes fails to fully exhibit its function as a clamp probe or becomes partly inactive, resulting in decreased efficiency in clamp formation, although such a phenomenon should not have occurred considering the high photoreactivity of the photoresponsive nucleic acid. This inactivation of the photoresponsive nucleic-acid probe not only decreases the efficiency of the photo-clamping method but also can cause problems in any of the applications of photoresponsive nucleic-acid probes.
An object of the present invention is to provide a means for preventing inactivation of a photoresponsive nucleic-acid probe.
The inventors of the present invention have conducted intensive research and, as a result, have found that inactivation of a photoresponsive nucleic-acid probe in the photo-clamping method occurs when the base sequence of the photoresponsive nucleic-acid probe is capable of forming a double-stranded section within the base sequence: in which case, the photoresponsive nucleic-acid probe containing a double-stranded section within the base sequence undergoes photo-crosslinkage, in other words, the photoresponsive nucleic-acid probe undergoes self-crosslinkage, and consequently loses its intended ability to form a double strand with a complementary strand and then undergo photo-crosslinkage.
This finding suggests that the inactivation can be suppressed, for example, by avoiding use of a base sequence capable of forming a double strand within the base sequence, in the first place. However, this approach ends up limiting the range of applications of the photoresponsive nucleic-acid probe, especially severely when use of a long base sequence is desired.
The inventors of the present invention have conducted further research to avoid this limitation from being imposed on the base sequence of a photoresponsive nucleic-acid probe. Thymine (T), which is a base to which a photoresponsive nucleic acid as a photoresponsive nucleic-acid probe is photo-linked, is known to maintain its ability to undergo photo-crosslinkage with a photoresponsive nucleic acid even when the thymine (T) has various modifications provided that double-strand formation with a complementary strand can still occur. Here, the inventors of the present invention have found that by substituting the C5 of thymine with a cyano group, which is an electron-withdrawing group, and converting the thymine into 5-cyano-2′-deoxyuridine (CNT), the speed of photo-crosslinkage with a photoresponsive nucleic acid becomes very low without affecting double-strand formation with a complementary strand. Thus, the present invention has now been completed. In other words, introduction of 5-cyano-2′-deoxyuridine (CNT) that is obtained by substituting the C5 of thymine with an electron-withdrawing cyano group can suppress inactivation of a photoresponsive nucleic-acid probe.
In addition to the suppression of inactivation caused by self-crosslinkage occurring within a photoresponsive nucleic-acid probe, the finding described above is also widely applicable to preventing unintended photo-crosslinkage of a thymine (T) or uracil (U) base that is present near a base sequence complementary to a base sequence present near a photoresponsive base, and consequent failure of intended photo-crosslinkage, and also to preventing overconsumption of a photoresponsive nucleic acid and a consequent decrease in efficiency and yield of target reaction.
The present invention subsumes the following, starting from (1).
(1) A method of suppressing photo-crosslinkage of a photoresponsive base having a photo-crosslinkable vinyl structure with a thymine (T) or uracil (U) base that is photo-crosslinkable to the photoresponsive base having a photo-crosslinkable vinyl structure, comprising:
substituting C5 of a pyrimidine ring of the thymine (T) or uracil (U) base with an R group (R is -CN or -CO-R1, and R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group)
(2) The method according to (1), wherein
the photo-crosslinkage is a reaction in which a photo-crosslink is formed between:
the photoresponsive base having a photo-crosslinkable vinyl structure, the photoresponsive base having a photo-crosslinkable vinyl structure being contained in a base sequence of a photoresponsive nucleic acid, and
the thymine (T) or uracil (U) base that is photo-crosslinkable to the photoresponsive base, the thymine (T) or uracil (U) base being contained in a base sequence fraction complementary to a base sequence fraction that is contained in the base sequence of the photoresponsive nucleic acid and is composed of 4 or more bases including the photoresponsive base (the base sequence fraction that is contained in the base sequence of the photoresponsive nucleic acid and is composed of 4 or more bases including the photoresponsive base is called a photoresponsive base sequence fraction) (the base sequence fraction complementary to the photoresponsive base sequence fraction is called a complementary base sequence fraction), and the complementary base sequence fraction being contained in a nucleic acid (a partially-complementary nucleic acid), and
in the complementary base sequence fraction contained in the partially-complementary nucleic acid, at least one constituent nucleotide containing the photo-crosslinkable T or U is replaced by a modified nucleotide of Formula (I):
(in Formula I,
R is -CN or -CO-R1,
R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group,
X forms a phosphate group together with O that is bonded to X in Formula I,
Y is a hydroxy group, and
Z is hydrogen or a hydroxy group), the modified nucleotide of Formula (I) being introduced to the complementary base sequence fraction by a phosphodiester bond, and as a result, photo-crosslinkage of the photoresponsive nucleic acid with the partially-complementary nucleic acid is suppressed.
(3) The method according to (1) to (2), wherein
the photoresponsive nucleic acid contains both the photoresponsive base sequence fraction and the complementary base sequence fraction as separate sequence regions within a molecule of the photoresponsive nucleic acid, the molecule of the photoresponsive nucleic acid being the same as a molecule of the partially-complementary nucleic acid, and
suppression of the photo-crosslinkage of the photoresponsive nucleic acid with the partially-complementary nucleic acid is achieved by suppressing self-crosslinkage within the photoresponsive nucleic acid caused by the photo-crosslinkage of the photoresponsive base with the photo-crosslinkable thymine (T) or uracil (U) base.
(4) The method according to any one of (1) to (3), wherein R in Formula (I) is -CN.
(5) The method according to any one of (1) to (4), wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a photoresponsive base having a 3-vinylcarbazole structure.
(6) The method according to any one of (1) to (5), wherein
the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of Formula (II):
(in Formula II, Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
X forms a phosphate group together with O that is bonded to X in Formula II,
Z is hydrogen or a hydroxy group), and
the photoresponsive base having a photo-crosslinkable vinyl structure is introduced into the photoresponsive base sequence fraction as a base portion of the modified nucleotide by a phosphodiester bond of the modified nucleotide.
the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of Formula (III):
(in Formula III,
Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic group, a monovalent group of a monocyclic or dicyclic, C6-C12 heterocyclic aromatic group, or a monovalent group of a formula:
(wherein Ra, R2, and R3 are independent of Ra, R2, and R3 as defined for Formula III and are selected from the groups given above as examples of Ra, R2, and R3 for Formula III),
R6 is a hydrogen atom, a methyl group, or an ethyl group,
Q1 forms a phosphate group together with O that is bonded to Q1 in Formula III, and
Q2 is a hydrogen atom), and
(8) The method according to (7), in which in Formula III, a backbone structure of Formula (IIIa):
is a D-threoninol structure of the formula:
an L-threoninol structure of the formula:
or a serinol structure of the formula:
(9) The method according to any one of (1) to (8), wherein every nucleotide containing T or U in the base sequence of the partially-complementary nucleic acid is substituted with the modified nucleotide of Formula (I).
The present invention further subsumes the following, starting from (11).
(11) A photoresponsive nucleic acid with suppressed self-crosslinking ability, comprising:
a photoresponsive base having a photo-crosslinkable vinyl structure, and
a modified nucleotide of Formula (I):
Z is hydrogen or a hydroxy group), in place of at least one constituent nucleotide containing photo-crosslinkable T or U.
(12) The photoresponsive nucleic acid with suppressed self-crosslinking ability according to (11), wherein R in Formula (I) is -CN.
(13) The photoresponsive nucleic acid with suppressed self-crosslinking ability according to (11) or (12), wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a photoresponsive base having a 3-vinylcarbazole structure.
(14) The photoresponsive nucleic acid with suppressed self-crosslinking ability according to any one of (11) to (13), wherein
the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of or Formula (III):
(16) The photoresponsive nucleic acid with suppressed self-crosslinking ability according to (15), in which in Formula III, a backbone structure of Formula (IIIa):
the photoresponsive nucleic acid with suppressed self-crosslinking ability comprises a base sequence fraction complementary to a base sequence fraction composed of 4 or more bases including the photoresponsive base (the base sequence fraction composed of 4 or more bases including the photoresponsive base is called a photoresponsive base sequence fraction) (the base sequence fraction complementary to the photoresponsive base sequence fraction is called a complementary base sequence fraction), and
the photo-crosslinkable T or U is T or U contained in the complementary base sequence fraction.
(18) The photoresponsive nucleic acid with suppressed self-crosslinking ability according to any one of (11) to (17), wherein every nucleotide containing T or U in the base sequence of the photoresponsive nucleic acid with suppressed self-crosslinking ability is substituted with the modified nucleotide of Formula (I).
According to the present invention, self-crosslinkage within a photoresponsive nucleic-acid probe used in the photo-clamping method or the like can be suppressed, and as a result, inactivation of the probe can also be prevented. Consequently, the photoresponsive nucleic-acid probe can be enhanced in its reaction efficiency (yield) with no particular limitation given on the type or the length of the base sequence. Furthermore, the present invention can prevent unintended photo-crosslinkage of a thymine (T) or uracil (U) base that is present near a base sequence complementary to a base sequence present near a photoresponsive base, and consequent failure of intended photo-crosslinkage, and can also prevent overconsumption of a photoresponsive nucleic acid and a consequent decrease in efficiency and yield of target reaction. Therefore, the present invention can enhance the range of applications of a photoresponsive nucleic acid.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the results of non-native PAGE comparing the photoreactivity of T with the photoreactivity of CNT.
FIG. 2 shows the results of non-native PAGE analysis on samples that underwent longer irradiation.
FIG. 3 shows HPLC charts and a graph showing the difference in photoreactivity between CNT and T.
FIG. 4 shows the scheme and the results of photoreaction occurred in an experiment that was carried out to study photo-crosslinkage of a CNVK-containing probe.
FIG. 5 shows the results of an experiment carried out to study the ability of CNT to suppress inactivation of a CNVK-containing probe.
FIG. 6 is the scheme of photoreaction occurred in an experiment that was carried out to study photo-crosslinkage of a CNVD-containing probe.
FIG. 7 is a graph showing the results of an experiment carried out to study photo-crosslinkage of a CNVD-containing probe.
In the following, the present invention will be described in detail referring to specific embodiments. The scope of the present invention, however, is not limited to these specific embodiments.
[Photo-Crosslinkage]
A photoresponsive base having a photo-crosslinkable vinyl structure undergoes highly selective photo-crosslinkage with thymine (T) or uracil (U) and cytosine, each of which is a pyrimidine base (Patent Document 1). The photo-crosslinking reaction is a photoreaction, and therefore proceeds extremely rapidly with high efficiency. The desirable solvent conditions and temperature conditions for the photo-crosslinking reaction can be selected from a wide range of conditions including physiological conditions. A photoresponsive base having a 3-vinylcarbazole structure, in particular, undergoes photoreaction, namely, [2+2] photocyclization reaction with a pyrimidine base with high efficiency to form a photo-crosslink.
This photo-crosslinkage also proceeds rapidly and efficiently even when the pyrimidine base has modification. Therefore, suppression of photo-crosslinkage by modifying the pyrimidine base while maintaining complementation necessary for base-pair formation has not been successfully achieved. The photo-crosslinkage also proceeds, for example, when the pyrimidine base is methylcytosine or pseudo uridine.
[Suppression of Photo-Crosslinkage]
Here, according to the present invention, by substituting the C5 of a pyrimidine ring of a thymine (T) or uracil (U) base that is photo-crosslinkable to the photoresponsive base having a photo-crosslinkable vinyl structure, with an R group (R is —CN or —CO—R1, and R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group), photo-crosslinkage can be suppressed while the resulting modified base with substitution does not lose but maintains its complementation necessary for base-pair formation. The suppression of photo-crosslinkage is achieved at an extremely remarkable level, which is indicated by the fact that the efficiency of photo-crosslinking reaction was suppressed to about 1% even under conditions (duration of irradiation) that allowed photo-crosslinking reaction of thymine (T) to proceed at an efficiency of greater than 90% (see the Example section).
[Double-Strand Formation Prior to Photo-Crosslinkage]
Photo-crosslinkage of the photoresponsive base with the thymine (T) or uracil (U) base occurs in the following way: prior to irradiation, a base sequence containing the photoresponsive base together with a base sequence containing the thymine (T) or uracil (U) base form a double strand based on their complementation and are consequently positioned so as to allow photo-crosslinking reaction to occur, and then upon irradiation, photoreaction proceeds well. In a preferred embodiment, a base sequence fraction that is contained in the base sequence of the photoresponsive nucleic acid containing the photoresponsive base and that contains the photoresponsive base (the base sequence fraction is called a photoresponsive base sequence fraction) is complementary to a base sequence fraction containing the thymine (T) or uracil (U) base (the base sequence fraction containing the thymine (T) or uracil (U) base is called a complementary base sequence fraction). In a preferred embodiment, either of the photoresponsive base sequence fraction and the complementary base sequence fraction is stable enough to form a double-stranded region together, and contains at least 4 bases, for example, preferably at least 5 bases, further preferably at least 6 bases, further preferably at least 7 bases, and further preferably at least 8 bases.
[Modified Base and Modified Nucleotide for Photo-Crosslinkage Suppression]
The modified base introduced in place of the photo-crosslinkable thymine (T) or uracil (U) base so as to suppress photo-crosslinkage (the modified base is called a modified base for photo-crosslinkage suppression) has the C5 of the pyrimidine ring substituted with an R group (R is —CN or —CO—R1, and R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group). A modified nucleotide containing the modified base as its base portion (a modified nucleotide for photo-crosslinkage suppression) is a modified nucleotide of Formula (I):
In Formula I, R is —CN or —CO—R1, preferably —CN (a cyano group). R1 can be any R1 provided that an electron-withdrawing R group is formed, and is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group, for example. Examples of this group include a C1-C3 alkyl group, a cyclohexyl group, a phenyl group, a benzyl group, a tolyl group, and a naphthyl group.
In Formula I, X forms a phosphate group together with O that is bonded to X in Formula I, Y is a hydroxy group, and Z is hydrogen or a hydroxy group.
[Synthesis of Modified Base and Modified Nucleotide for Photo-Crosslinkage Suppression]
The modified base for photo-crosslinkage suppression and the modified nucleotide for photo-crosslinkage suppression can be synthesized by a known means. First, an amidite thereof is synthesized, and then by a known means using a DNA synthesizer or the like, a nucleic acid to which the modified base for photo-crosslinkage suppression is introduced in place of the photo-crosslinkable thymine (T) or uracil (U) base (the nucleic acid is called a modified nucleic acid) can be produced. When desired, the nucleic acid to which the modified base for photo-crosslinkage suppression is introduced in place of the photo-crosslinkable thymine (T) or uracil (U) base (modified nucleic acid) can be produced by first producing a nucleic acid containing the thymine (T) or uracil (U) base or another modified base (modified nucleic acid) and then subjecting the resultant to modification reaction to convert the base into the modified base for photo-crosslinkage suppression.
[Photoresponsive base having photo-crosslinkable vinyl structure]
The photoresponsive base having a photo-crosslinkable vinyl structure is preferably a photoresponsive base having a 3-vinylcarbazole structure, and further preferably a base portion of a modified nucleotide of Formula (II):
In Formula II, Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
Z is hydrogen or a hydroxy group.
In a preferred embodiment, Ra in Formula II is a cyano group, an amido group, a carboxy group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amido group, a carboxy group, an alkoxycarbonyl group, or hydrogen, and further preferably a cyano group, an amido group, a carboxy group, or an alkoxycarbonyl group. The alkoxycarbonyl group that can be used is preferably a C2-C7 alkoxycarbonyl group, further preferably a C2-C6 alkoxycarbonyl group, further preferably a C2-C5 alkoxycarbonyl group, further preferably a C2-C4 alkoxycarbonyl group, further preferably a C2-C3 alkoxycarbonyl group, and particularly preferably a C2 alkoxycarbonyl group.
In a preferred embodiment, R2 and R3 in Formula II are independently a cyano group, an amido group, a carboxy group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amido group, a carboxy group, an alkoxycarbonyl group, or hydrogen, and further preferably a cyano group, an amido group, a carboxy group, or an alkoxycarbonyl group. The alkoxycarbonyl group that can be used is preferably a C2-C7 alkoxycarbonyl group, further preferably a C2-C6 alkoxycarbonyl group, further preferably a C2-C5 alkoxycarbonyl group, further preferably a C2-C4 alkoxycarbonyl group, further preferably a C2-C3 alkoxycarbonyl group, and particularly preferably a C2 alkoxycarbonyl group.
In a preferred embodiment of the present invention, the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of Formula (III):
In Formula III,
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic compound, a monovalent group of a monocyclic or dicyclic, C6-C12 heterocyclic aromatic compound, or a monovalent group of a formula:
(wherein Ra, R2, and R3 are independent of Ra, R2, and R3 as defined for Formula III, and are selected from the groups given above as examples of Ra, R2, and R3 for Formula III),
Q2 is a hydrogen atom.
Ra, R2, and R3 in Formula III can be independently the same as Ra, R2, and R3 as defined for Formula II.
For example, R4 in Formula III can be selected from the following groups (each wavy line indicates where the free valency is located).
In a preferred embodiment, in Formula III, the backbone structure of Formula (IIIa):
is a D-threoninol structure of a formula:
an L-threoninol structure of a formula:
or a serinol structure of a formula:
The sugar backbone portion of the modified nucleotide of Formula III is not of a ribose (or deoxyribose) structure as in a natural nucleotide or the modified nucleotide of Formula II, but is replaced by the backbone structure of Formula IIIa. Therefore, the modified nucleotide of Formula III can also be called an artificial photoresponsive nucleotide analog. Even though it has such a different backbone structure, the modified nucleotide of Formula III unexpectedly acts as a photoresponsive base as far as its incorporation into a nucleic acid and its photoresponsivity are concerned. Based on this finding, the inventors of the present invention have already filed a patent application in Japan (Japanese Patent Application No. 2013-70381).
[Suppression of Self-Crosslinkage]
The suppression of photo-crosslinkage according to the present invention is useful, needless to say, when the photoresponsive base and the thymine (T) or uracil (U) base are contained in separate nucleic acid molecules and the photo-crosslinkage occurs between these separate nucleic acid molecules. The suppression of photo-crosslinkage according to the present invention is also useful, in particular, when the photoresponsive base and the thymine (T) or uracil (U) base are contained in the same nucleic acid molecule and self-crosslinkage occurs within the nucleic acid molecule. For example, a photoresponsive nucleic-acid probe used in the photo-clamping method can have a double-stranded section formed within the nucleic acid molecule, depending on the base sequence of the nucleic acid molecule. In this case, when the double-stranded section contains both a photoresponsive base and a thymine (T) or uracil (U) base positioned in a way that they can undergo photo-crosslinkage, the photoresponsive nucleic-acid probe undergoes intramolecular self-crosslinkage and consequently fails to participate in intended photo-crosslinking reaction, resulting in a significant decrease in the efficiency of use of the probe (or in the yield). By suppressing self-crosslinkage within the photoresponsive nucleic-acid probe, loss of the probe is prevented and excellent efficiency of use (or high yield) is obtained. In other words, in a preferred embodiment, the photoresponsive base sequence fraction and the complementary base sequence fraction are contained in the same nucleic acid molecule and self-crosslinkage therebetween is suppressed.
[Substitution of Every Thymine (T) or Uracil (U) Base]
According to the present invention, by substituting at least one photo-crosslinkable thymine (T) or uracil (U) base in the nucleic acid molecule with the modified base for photo-crosslinkage suppression, photo-crosslinkage of the thymine (T) or uracil (U) base can be suppressed. Therefore, substitution of at least one base is subsumed within the scope of the present invention.
The nucleic acid substituted with the modified base for photo-crosslinkage suppression of the present invention is superior to an unsubstituted nucleic acid containing a thymine (T) or uracil (U) base because the Tm value thereof does not fluctuate and the ability thereof to form a double strand is maintained. In other words, substitution with the modified base for photo-crosslinkage suppression of the present invention does not affect the ability thereof to form a double strand, and therefore there is no need to limit the number of substitution to the minimum and instead every thymine (T) or uracil (U) base contained in a target nucleic acid molecule can be substituted with the modified base for photo-crosslinkage suppression. Substitution of every thymine (T) or uracil (U) base is advantageous because no analysis is required on the structure of the complementary strand and substitution can be carried out uniformly across the complementary strand to suppress any undesired photo-crosslinkage. Substitution of every thymine (T) or uracil (U) base is particularly advantageous, for example, when producing photoresponsive nucleic-acid probes having many different base sequences.
[Reaction Conditions in Photo-Crosslinkage Suppression]
According to the present invention, photo-crosslinkage can be suppressed under conventionally known conditions for photo-crosslinkage. For example, the light for irradiation to cause photo-crosslinkage usually has a wavelength within the range from 350 nm to 380 nm, preferably has a wavelength within the range from 360 nm to 370 nm, further preferably has a wavelength of 366 nm, and particularly preferably is laser light having a single wavelength of 366 nm. The suppression of photo-crosslinkage can be achieved under any of these irradiation conditions.
The present invention will be described in detail referring to examples. The scope of the present invention, however, is not limited to these examples.
[Synthesis of CNVK-Containing ODN]
Synthesis of an amidite of a nucleotide (CNVK) of a formula:
3-cyanovinylcarbazole (CNVK)
was carried out according to Scheme 1 below. Synthesis was carried out according to the procedure disclosed in Patent Document 1 (International Publication No. WO 2009/066447).
In the same manner, an amidite of a nucleotide (CNT) of a formula:
5-cyano-2′-deoxyuridine (CNT)
was synthesized.
The resulting amidites of the artificial photoresponsive nucleic acids, namely, 3-cyanovinylcarbazole nucleotide (CNVK) and 5-cyano-2′-deoxyuridine (CNT) were made to be 100 mM with the use of acetonitrile, and an ABI3400 was used to synthesize an ODN. The sequence of the resulting ODN is shown in Table 1. Following synthesis, deprotection was carried out with a 28% aqueous ammonia solution at 55° C. for 8 hours. Subsequently, purification was carried out with HPLC, followed by mass spectroscopy, which confirmed that the intended sequence was obtained.
ODNs used in experiment
Number Sequence (5′-3′) of bases
ODN 1 TGCACNVKACGT (SEQ ID NO: 4) 9 ODN 2 ACGTGTGCA (SEQ ID NO: 5) 9 ODN 3 ACGTGCNTGCA (SEQ ID NO: 6) 9 ODN 4 GTACNVKAGAGTGTA (SEQ ID NO: 1) 13 ODN 5 GCNTACNVKAGAGCNTGCNTA (SEQ ID NO: 2) 13 ODN 6 AGAGTACACTCTATACTGAG (SEQ ID NO: 3) 20
[Analysis of Photoreactivity of CNVK and CNT]
A buffer (100 mM NaCl, 50 mM sodium cacodylate) containing 20 μM of ODN1 and 20 μM of ODN2 or 20 μM of ODN3 was heated at 90° C. for 5 minutes, followed by annealing with the temperature being slowly lowered to 4° C. Subsequently, a UV-LED irradiator was used to perform irradiation of UV at 366 nm at 4° C., and non-native PAGE analysis was carried out to confirm that a photo-crosslinked product had been formed by irradiation. The results are shown in FIG. 1.
FIG. 1 shows the results of non-native PAGE comparing the photoreactivity of T with the photoreactivity of CNT. As for the lanes in FIG. 1, M: 10 DNA Ladder Maker, Lanes 1 to 5: T as Target base, Lanes 6 to 10: CNT as Target base. Duration of irradiation was 0 s (second), 0.1 s, 0.5 s, 1 s, and 2 s. ODN1 and ODN2 hybridized with each other to form a double strand, and upon irradiation, CNVK underwent photo-crosslinkage with a photoresponsive base (Target base) T facing and complementary to the base adjacent to CNVK on the 5′ side. Electrophoresis detected a crosslinked product. The sequence of ODN1 and the sequence of ODN3 also hybridized with each other to form a double strand, and in this case, the base (Target base) facing and complementary to the base adjacent to CNVK on the 5′ side was CNT.
When ODN1 and ODN2 were paired, in other words, when the Target base was T, irradiation for 2 seconds resulted in near disappearance of a band attributable to the starting molecule, 9 mer. When ODN1 and ODN3 were paired, in other words, when Target was CNT, no band attributable to a crosslinked product was confirmed even after irradiation for 2 seconds. These results have proven that reactivity is significantly different between when the base to which CNVK crosslinked to is T and when the base to which CNVK crosslinked to is CNT. In order to investigate whether crosslinking reaction does not proceed at all or proceeds slowly when the base to which CNVK crosslinked to is CNT, another non-native PAGE analysis was carried out on samples that had undergone longer irradiation. The results are shown in FIG. 2.
FIG. 2 shows the results of non-native PAGE analysis of samples that underwent longer irradiation. As for the lanes, the lane M is attributed to 10 bp DNA Ladder Maker, and the rest are attributed to duration of irradiation of 0 second, 1 second, 5 seconds, 10 seconds, 30 seconds, 60 seconds, 120 seconds, and 300 seconds.
FIG. 2 shows that when ODN1 and ODN3 were paired, bands probably attributable to a photo-crosslinked product were barely confirmed for samples that had been irradiated for significantly extended periods of time. These results have proven that CNT in which the C5 of thymine is substituted with a cyano group is crosslinked to CNVK at a speed much lower than thymine (T) is.
For quantitative discussion, HPLC analysis was carried out and the decrement of ODN3 was used to calculate a photo-crosslinking rate. The results are shown in FIG. 3.
FIG. 3 includes FIG. 3(A), which shows the results of HPLC analysis regarding crosslinking reaction between ODN1 and ODN3. FIG. 3(A) shows charts obtained after irradiation for 0 s (second), 0.1 s, 0.5 s, 1 s, 2 s, and 5 s, with the abscissa indicating retention time (minute). FIG. 3 also includes FIG. 3(B), which is a graph comparing the photoreactivity of ODN2 (T) with the photoreactivity of ODN3 (CNT). The abscissa indicates duration of irradiation (second), and the ordinate indicates the photo-crosslinking rate (%). In FIG. 3(B), the upper approximate curve is drawn for ODN2 (T), and the lower approximate curve is drawn for ODN3 (CNT).
These HPLC results have also proven that mere several seconds of irradiation allowed almost no photo-crosslinking reaction to proceed between CNT and CNVK. The photo-crosslinking rate calculated from the decrement of ODN3 occurred upon irradiation was about 1% when Target was CNT and the duration of irradiation was 1 second, compared to 90% or higher when Target was T and the duration of irradiation was 1 second. Curve fitting was carried out to obtain an approximate curve, which has proven that photo-crosslinking reaction proceeded slowly when Target was CNT, at about 1/80 the speed when Target was thymine.
[Suppression of Inactivation of CNVK-Containing Probe]
CNVK is highly photoresponsive, and therefore may undergo crosslinkage even when the double strand structure is only temporarily formed and not very stable. Formation of a self-crosslinked structure, in particular, is considered to cause inactivation of a CNVK-containing probe. Based on the findings above that CNT is poorly photoresponsive to CNVK, an experiment was carried out to study suppression of inactivation of a CNVK-containing probe.
Prior to a series of experiments, screening was first carried out to select a self-crosslinkable base sequence. As a result, determination was made to use the ODN4 sequence, and the experiments followed.
A buffer (100 mM NaCl, 50 mM sodium cacodylate) containing 20 μM of Probe ODN (ODN4 or ODN5) and 20 μM of ODN6 was heated at 90° C. for 5 minutes, followed by annealing with the temperature being slowly lowered to 25° C. As for Lanes 5 and 6, sample preparation was followed by heating at 90° C. for 5 minutes, and then the resultant was immediately transferred to 25° C. for rapid quenching. As for Lanes 7 and 8, Probe ODN at 20 μM and ODN6 at 4 μM were used, where the contents of Probes were 5 times greater than the content of Target. These samples were irradiated with UV at 366 nm with the use of a UV-LED irradiator, at 25° C. for 10 seconds. Then, non-native PAGE analysis was performed. The procedure and the results are shown in FIG. 4.
FIG. 4 shows the scheme and the results of photoreaction occurred in an experiment that was carried out to study photo-crosslinkage of a CNVK-containing probe. FIG. 4(A) is a descriptive view of the scheme of photoreaction of the CNVK-containing probe undergoing photo-crosslinkage. FIG. 4(B) shows the results of non-native PAGE carried out in the experiments. As for the lanes in FIG. 4(B), Lane M is attributable to 10 bp DNA Ladder Maker, Lanes 1 and 2 are attributable to Probe alone, Lanes 3 and 4 are attributable to Probe and Target, Lanes 5 and 6 are attributable to Probe and Target after rapid quenching, and Lanes 7 and 8 are attributable to the case where the contents of Probes were 5 times greater than the content of Target (5 equivalents). Lanes 2, 4, 6, and 8 received irradiation, and Lanes 1, 3, 5, and 7 received no irradiation.
After irradiation, the CNVK-containing probe used here yielded a new band slightly off, to the low-molecular-weight side, the band attributable to the starting molecule. This new band did not appear in the case of no irradiation, appeared when the CNVK-containing probe (ODN4) alone was added, and appeared separately from the band attributable to ODN4 when electrophoresis was carried out under non-native conditions. From these and other results, the new band was probably attributable to a self-crosslinked product resulting from intramolecular photo-crosslinkage within the CNVK-containing probe (ODN4).
Another experiment was carried out in the same manner but using a probe in which every T contained in ODN4 was substituted with CNT. The results are shown in FIG. 5.
FIG. 5 shows the results of an experiment carried out to study suppression of inactivation of a CNVK-containing probe by CNT. FIG. 5(A) shows the results with an ordinary probe, and FIG. 5(B) shows the results with a probe substituted with CNT. As for the lanes, Lane M is attributable to 10 bp DNA Ladder Maker, Lanes 1 and 2 are attributable to Probe alone, Lanes 3 and 4 are attributable to Probe and Target, Lanes 5 and 6 are attributable to Probe and Target after rapid quenching, and Lanes 7 and 8 are attributable to the case where the contents of Probes were 5 times greater than the content of Target (5 equivalents). Lanes 2, 4, 6, and 8 received irradiation, and Lanes 1, 3, 5, and 7 received no irradiation.
As shown in the results, substitution of T in the CNVK-containing probe (ODN4) with CNT resulted in no band confirmed attributable to a self-crosslinked product and a band noticeably appeared attributable to a crosslinked product. Thus, by substituting T contained in the CNVK-containing probe with CNT, only the self-crosslinkage within the CNVK-containing probe (ODN5) can be suppressed with no inhibition occurring on double-strand formation or on photo-crosslinking reaction with ODN6 targeted by the CNVK-containing probe, and, as a result, inactivation of the probe can be suppressed.
Within the CNVK-containing probe, base sequence sections that are self-crosslinkable are very limited. However, in such a case that various sequences present in living organisms are targeted or a long-chain probe is used, undesired self-crosslinkage can occur. By substituting T contained in the CNVK-containing probe with CNT as described above, such undesired self-crosslinkage can be suppressed. Substitution of T with CNT does not impair the ability of the probe to form a double strand and to undergo photo-crosslinking reaction, and therefore every T can be substituted with CNT. For example, simulation can be carried out to predict a structure that is self-crosslinkable to a certain extent, and then only the Ts contained in the structure predicted to be self-crosslinkable can be substituted with CNTs. These findings allow a CNVK-containing probe to be used in a wider range of applications and to be applied to a wider range of sequences.
[Tm Value]
Between the Tm value for the pair of ODN1 and ODN2 and the Tm value for the pair of ODN1 and ODN3, no measurable difference was observed. This indicates that the ability to form a double strand was maintained after substitution of T with CNT.
[Synthesis of CNVD-Containing ODN]
A nucleotide analog (CNVD) of the following formula was synthesized according to Scheme 2. Subsequently, an amidite of the nucleotide analog (CNVD) was synthesized, which was then used to synthesize ODN in the same manner as in the case of CNVK above, for use in analysis of photoreactivity with CNT.
[Analysis of Photoreactivity Between CNVD and CNT]
An experiment was carried out in the same manner as in the experiment described above where CNVK was used, except that ODN containing CNVD instead of CNVK was used, in order to compare photoreactivity with CNT to photoreactivity with T. FIG. 6 is the scheme of photoreaction occurred in an experiment that was carried out to study photo-crosslinkage of a CNVD-containing probe. FIG. 7 is a graph showing the results of an experiment carried out to study photo-crosslinkage of a CNVD-containing probe. As indicated by the results shown in FIG. 7, use of CNT instead of T suppressed photo-crosslinking reaction of CNVD to about 15% (comparison was made after 1 second of irradiation).
According to the present invention, self-crosslinkage within a photoresponsive nucleic-acid probe used in the photo-clamping method or the like can be suppressed, and as a result, inactivation of the probe can be prevented. Therefore, the present invention is industrially useful.
1. A double stranded nucleic acid, comprising:
a first oligonucleotide, the first oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
wherein, in Formula II,
Ra is a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R2 and R3 are independently a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
X forms a phosphate group together with O that is bonded to X,
Z is a hydrogen atom or a hydroxyl group;
wherein, in Formula III,
Ra is a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic group, a naphthyl group, an indolyl group, a benzoimidazolyl group, a benzothiophenyl group, or a monovalent group of a formula:
wherein Ra, R2, and R3 are independent of Ra, R2, and R3 as defined for Formula III and are selected from the groups given above as examples of Ra, R2, and R3 for Formula III,
Q1 forms a phosphate group together with O that is bonded to Q1, and
Q2 is a hydrogen atom, and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into a base sequence fraction as a base portion of the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond, and
a second oligonucleotide, the second oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
wherein, in Formula I:
Z is a hydrogen atom or a hydroxy group, and
wherein the first oligonucleotide and the second oligonucleotide are hybridized.
2. The double stranded nucleic acid according to claim 1, wherein R in Formula (I) is a cyano group.
3. The double stranded nucleic acid according to claim 2,
wherein the first oligonucleotide comprises 4 or more bases.
5. The double stranded nucleic acid according to claim 4, wherein the modified nucleotide having the structure corresponding to the monomer of Formula (I) in the second oligonucleotide is at every position complementary to the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) in the first oligonucleotide.
wherein the first oligonucleotide contains the modified nucleotide having the structure corresponding to the monomer of Formula (I), and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (I) replaces at least one constituent nucleotide which is the photo-crosslinkable thymine (T) or uracil (U).
9. The double stranded nucleic acid according to claim 6, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.
10. The double stranded nucleic acid according to claim 9,
wherein either: 1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or 2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.
12. The double stranded nucleic acid according to claim 11,
wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom, and
15. The double stranded nucleic acid according to claim 1, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.
21. A single stranded oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
R is -CO-R1,
R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 unsubstituted hydrocarbon group,
Z is a hydrogen atom or a hydroxy group.
22. The single stranded oligonucleotide according to claim 21, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.
23. A single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability comprising:
a first base sequence, the first base sequence comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic group, a monovalent group of a monocyclic or dicyclic, naphthyl group, indolyl group, benzoimidazolyl group, benzothiophenyl group or a monovalent group of a formula:
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into a base sequence fraction as a base portion of the modified nucleotide of having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond,
a second base sequence, the second base sequence comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
24. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein R in Formula (I) is a cyano group.
wherein R is a cyano group, and Z is a hydrogen atom.
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).
wherein R is a cyano group, and Z is a hydrogen atom,
wherein either: 1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or 2) wherein, in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.
44. A method of hybridizing nucleic acid sequences, comprising:
performing a hybridizing reaction of a second oligonucleotide with a first oligonucleotide by mixing a first base sequence fraction including the first oligonucleotide with a second base sequence fraction including the second oligonucleotide, the second oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
Z is a hydrogen atom or a hydroxy group,
wherein the modified nucleotide having a structure corresponding to the monomer of Formula (I) replaces at least one constituent nucleotide which is the photo-crosslinkable thymine (T) or uracil (U),
the first oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into the second base sequence fraction as a base portion of the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond, and
wherein the first oligonucleotide is at least partially complementary to the second oligonucleotide sequence fraction.
45. The method of hybridizing nucleic acid sequences according to claim 44,
46. The method of hybridizing nucleic acid sequences according to claim 45, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.
wherein either: 1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or 2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, and R6 is a methyl group, and Q2 is a hydrogen atom.
wherein, either 1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or 2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2is a hydrogen atom.
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Assignee: JAPAN SCIENCE AND TECHNOLOGY AGENCY (Kawaguchi)
Inventors: Kenzo Fujimoto (Nomi), Shigetaka Nakamura (Nomi)
Primary Examiner: Eric Olson
Current U.S. Class: Trivalent Phosphorus Compound Utilized (536/25.34)
International Classification: C07H 19/06 (20060101); C07H 19/04 (20060101); C07H 21/04 (20060101); C07H 19/073 (20060101); C07H 19/10 (20060101); C07H 19/044 (20060101);


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text:
Die Düna (, , , ) ist ein in die Ostsee mündender, 1020 km langer Strom.

Der russische Name  bedeutet wörtlich übersetzt "Westliche Dwina" zur Unterscheidung von der Nördlichen Dwina.

Verlauf
Der Fluss beginnt in Russland auf den Waldaihöhen südlich von Bobrowez, Andreapolski Rajon, Oblast Twer nahe den Wolgaquellen.

Die Kleine Düna () fließt bei  () aus dem Korjakino-See () etwa vier Kilometer bis in die  (). Der Durchfluss durch die Seen für circa 20 km wird bereits als Westliche Düna () angesehen. Beim  () beginnt der eigentliche Flusslauf der Düna.

Sie fließt zunächst nach Südwesten. Bei  () ist die Flussmitte für zwei Kilometer Grenze zu Belarus und heißt jetzt Dswina (). Nach Wizebsk () setzt sie sich entlang dem Belarussischen Höhenrücken in westlichen Richtungen durch das nördliche Belarus fort.

Neben  () zweigt das Kanalsystem Beresina (, ) südlich ab. Früher war es eine Verbindung zum Dnjepr und somit Teil der Verbindung von der Ostsee zum Schwarzem Meer.

Bei  wird die Daugava für 17 km zum Grenzfluss und fließt weiter durch Lettgallen, das südöstliche Gebiet von Lettland, durch Daugavpils (deutsch Dünaburg) und danach nach Nordwesten zum Rigaischen Meerbusen, wo sie im Rigaer Stadtteil Daugavgrīva (deutsch Dünamünde) in die Ostsee mündet.

Die Gesamtlänge liegt, je nach Berechnungsart, zwischen 1005 und 1020 km. Die Länge auf lettischem Territorium beträgt 357 km, in Belarus 335 km.

Nebenflüsse
Anmerkung: Die Sortierung erfolgt jeweils flussabwärts.

Wortherkunft
Der Fluss taucht als Dina, Tina, Tuna, Veina oder Dyna zuerst in Wikingersagas und der Nestorchronik auf. Die livische Bezeichnung ist Veina oder Ven. Dvna, eine latinisierte Form des deutschen Namens Düna, wurde in handschriftlichen mittelalterlichen Dokumenten verwendet und ist noch auf Landkarten des 17. Jahrhunderts zu finden.

Die lettische und litauische Bezeichnung Daugava wurde von Philologen (August Bielenstein, Ernest Blese, Jānis Endzelīns u. a.) auf den Wortstamm "daudz" und "ūdens" zurückgeführt und bedeutete ursprünglich etwa "großes Wasser" oder "starker Strom".
Nach Ansicht des Philologen Konstantīns Karulis war in der lettischen Sprache ursprünglich der Wortstamm Dyna gebräuchlich. Er wurde zwischen dem 14. und dem 17. Jahrhundert schrittweise von der heutigen Bezeichnung verdrängt.

In lettischen Dainas wird der Fluss unter anderem als "Mütterchen Düna" () und als "Schicksalsfluss" besungen.

Geschichte

Die Düna war seit dem vierten Jahrtausend v. Chr. Siedlungsgebiet indoeuropäischer Zuwanderer, aus denen sich im Laufe der Zeit ostbaltische Stämme formierten.

Im ersten Jahrhundert n. Chr. wird in der antiken Schrift Geographike Hyphegesis des Claudius Ptolemäus ein Fluss namens Rhubon ( oder Rhudon) erwähnt, der die Düna meint oder die Memel.

Der Fluss war seit alter Zeit ein bedeutender Handelsweg, auf dem man ohne große Schwierigkeiten in die Flusssysteme der Wolga und des Dnepr gelangen konnte.

Spätestens seit dem achten Jahrhundert war die Düna eine der Routen der Waräger (Wikinger) auf dem "Weg von den Warägern zu den Griechen" ans Schwarze Meer. Wichtige Burgen waren unter anderem Grobin, Polazk und Wizebsk.

Seit dem neunten Jahrhundert sind an der Düna Kriwitschen um Polozk, seit dem 13. Jahrhundert die baltischen Stämme der Semgallen, Lettgallen und Selonen bekannt, aus denen sich in den folgenden Jahrhunderten das Volk der Letten entwickelte.

Im Mittelalter wurden die Handelswaren aufgrund des niedrigen Flussbetts und der vielen Stromschnellen bei Dünamünde von den größeren Schiffen auf Flöße oder Strusen für die Fahrt auf dem Fluss verladen.

Mit der Union von Wilna 1561 wurde der Fluss zu einer politischen und kulturellen Grenze.

Im 16. Jahrhundert änderte sich auch das Flussbett im Mündungsbereich. Der alte Abfluss, die Vecdaugava, versandete daraufhin.

Im Juli 1701 gab es im Rahmen des Großen Nordischen Krieges die Schlacht an der Düna, die in Krämershof () nördlich von Rīga (jetzt im Stadtgebiet) ausgetragen wurde.

Seit 1913 plante die "Riga-Cherson-Kanalkommission", ein Zusammenschluss von Rigenser Kaufleuten und Industriellen, einen Kanal zwischen Witebsk an der Düna und Orscha am Dnepr, der beide Ströme und so Riga mit der Hafenstadt Cherson am Schwarzen Meer verbinden sollte. Dabei waren auf der Düna sechs Staustufen mit je einem Wasserkraftwerk vorgesehen, u. a. bei Pļaviņas (dt. Stockmannshof), bei Ķegums (dt. Keggum) und bei Doles sala (dt. Dahlen) / Salaspils (dt. Kirchholm). Infolge des Ersten Weltkrieges, durch den das Kaiserreich Russland zerfiel und Lettland unabhängig wurde, zerschlug sich das Vorhaben des Kanal- und Wasserkraftwerkbaus.

Verkehr

Die Düna ist im russischen Abschnitt nur für den Bootsverkehr nutzbar. Bei ausreichendem Wasserstand ist sie ab Belarus schiffbar bis CEMT I, ab Jekabpils auch bis CEMT III. Im Mündungsbereich ist ab der Vanšu-Brücke in Riga der Seeverkehr möglich. Wegen einiger Sperrwerke ist ein durchgehender Schiffsverkehr nicht mehr möglich.

Im 19. Jahrhundert bestand eine Verbindung zwischen der Ostsee und Düna, via dem Nebenfluss Ula, dem Kanalsystem Beresina zum Dnjepr und bis in das Schwarze Meer. Durch den Ausbau der Bahn- und Straßenverbindungen verlor der Wasserweg an Bedeutung. Die wasserbautechnischen Anlagen verfielen. Diese Strecke ist nur noch teilweise für den Ausflugs- und Sportbootverkehr befahrbar.

Sehr beliebt bei Wassersportlern ist der Naturpark "Daugavas Loki" (Daugava-Bögen) zwischen Kraslava und Krauja.

Stauseen und Wasserkraftwerke
Was die junge Republik Lettland von den Planungen der "Riga-Cherson-Kanalkommission" verwirklichte, war der Bau des Stausees und des Wasserkraftwerkes bei Ķegums (Ķeguma hidroelektrostacija, meist abgekürzt: Ķeguma HES) von 1936 bis 1940. Der zweite Stausee mit Wasserkraftwerk wurde zur sowjetischen Zeit  – trotz heftiger Proteste der lettischen Bevölkerung  – von 1959 bis 1968 bei Pļaviņas errichtet (Pļaviņu hidroelektrostacija, meist abgekürzt: Pļaviņu HES). Der dritte lettische Stausee mit Wasserkraftwerk bei Salaspils nahe Riga (Rīgas hidroelektrostacija, meist abgekürzt: Rīgas HES) wurde 1974 fertiggestellt.

Die Proteste von Naturschützern gegen ein in den 1980er Jahren geplantes viertes Stauwerk bei Dünaburg bezeichnen den Beginn einer nationalen Bewegung, die 1990/91 zur Wiederherstellung der Unabhängigkeit Lettlands führte.

Am 30. Juni 2017 wurden ein Wasserkraftwerk und eine Schleuse etwa acht Kilometer stromaufwärts von Witebsk () in Betrieb genommen. Die vier Turbinen haben eine Gesamtleistung von 40 MW, die geplante jährliche Stromerzeugung wird mit 138 Mio. kWh angegeben. Die erzeugte Energie wird in das 110-kV-Netz übertragen.

Weblinks

Einzelnachweise

Fluss in der Oblast Smolensk
Fluss in der Wizebskaja Woblasz

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Design and Speed: Stuttgart's Exquisite Mercedes and Porsche Museums
October 20, 2017 by Tom Bentley4 Comments
For a portion of the population, a car is just a utilitarian thing: go from Point A to Point B, hope you don't run out of gas, and wash it every six months or so. But for some people, myself included, cars are an exuberant expression of ingenuity, excitement and progress. The automotive industry and its dynamic productions are colorful parts of the pageant of world history. And if you want to see some remarkably varied, thoughtful and outright thrilling accounts of that history, you must visit the Mercedes and Porsche museums in Stuttgart, Germany.
Both museum buildings are architectural masterworks of their own, befitting the housing of examples of extravagant vehicle design and power. Let's start with the Mercedes-Benz museum, which is patterned after the double helix of a DNA spiral, though to me it looked like a massive, whimsical layer cake of aluminum and glass. The surrounding grounds are appropriately stately, with the iconic Mercedes star presiding over all—seeing the building from a distance pulls your eyes like a magnet.
But it's inside where your heart begins to accelerate. But it will accelerate slowly, in parallel with Gottlieb Daimler's first efforts in developing the internal combustion engine and Carl Benz's work in putting that infernal thing on (three) wheels. The historical pathways of that development are part of the genius of the museum's design: There are seven Legend rooms, moving chronologically through Mercedes-Benz history, which dates back 130 years—and zooms through all its gears to the electric Mercedes-Benzes of today.
A Walk Through Automotive Time
Visitors walk down the seven Legend levels on a wide, circular ramp: cars of the period on the right, and images and exhibits of company history on the left. Very impressive to me were the interspersed world history exhibits, text and images, that were also part of the ramp wall art, and that correlated in time with the automotive materials. Thus you might see an image of the Beatles on one wall and some gleaming 60s Mercedes cars on the other side.
In concert with the Legend sections are Collection rooms of thematically-aligned vehicles, including buses, taxis, ambulances and celebrity cars. A well-designed audio tour (several languages) lets you pause at many of the gleaming machines and get a capsule history of their production and highlights. There's also a gallery set aside for temporary special collections.
There was something tremendously satisfying from seeing the first examples of the engines and rudimentary vehicles on the exhibit's top floor and then descending into the future, noting the captivations of the contours, sweeps and flourishes of luxury and style in succeeding years. I love the fluidity of the 1930s and 40s vehicles, and of course the legendary 1954 300SL Gull Wing, but admit to some affection for the humble 1980s 380SLs, since I had an '81 that I admired for its sturdy vigor.
Going Vertical with the Vehicles
However, my 380 would have been like a chained rock when compared to the rocket ships alleged to be cars in the lower level. Mercedes picked up more than a few racing trophies in its time, and there are some sleek, dangerous looking rides in this zone, which is highlighted by a vertical wall mounted by these snarling beasts of horsepower. Spectacular stuff. (And by the way, there were many people there when I visited, but it never seemed crowded. The exhibit spaces are well designed, and allow for lots of stopping and gawking room.)
Visitors can look into the working environment of Mercedes designers in the Fascination of Technology section, which shows research vehicles and exhibits on the design process. Nearby are some examples of experimental vehicles, and goodness, you will wonder what the designers had for breakfast. The nine-level building includes a beautiful atrium, a restaurant and a gift shop. It's an easy place to stay a long while—you can see why more than 800,000 people come to this museum every year. But even if you ask politely, they won't let you drive the Gull Wing.
Let Those Horses Run!
I loved many of the giant chariots at Mercedes, the big, dignified touring cars, so beautiful and stately. But when I went to the Porsche museum, another word came to mind: sexy. These low-slung, sleek, rounded rockets are sensual, and carried that sensuality even from the early days. As with the Mercedes museum, all that sexy is housed very nicely: the main building is a sculpted monolithic plane that half-floats in the air, hoisted on V-shaped columns. A strikingly nice touch is the three-columned, 80-foot-tall sculpture that boasts a version of the legendary 911 on each of its stelae. Bravo!
But it's inside where you'll really start to salivate, because that's where those low-slung beasts await you. Just to prime the thought carburetor, you should know that Ferdinand Porsche, the clever engineer behind the classic designs, designed the first gasoline-electric hybrid, way back in 1901, and electric cars as far back as 1896.
One pleasure of the Porsche museum is seeing some examples of these breakthrough engines and early vehicles, which stamp in your mind some of the shapes of more contemporary Porsches to come. You can see so much in the first prototype of the Porsche 356, from 1948, that graces the showroom. (My ideal Porsche in my youth was the beguiling 356 Speedster, though I later hankered after a more fire-breathing modern version. But I'm back to wanting a Speedster again.)
Fasten Your Seat Belt
Like with the Mercedes hosts, you'll be offered a digital audio device to get your facts straight about many of the cars you'll see on the tour. I even liked seeing the exhibits of the gears and camshafts and other engine parts, though working on engines was never my thing. But admiring the sweetly sloping bodies of the 60s and 70s vehicles is my thing—as well as being shocked out of my nostalgic reverie by the aggressive noses and flared fenders of the racing machines. Porsche has a sterling history of thrashing their opponents on the raceways, and there are some cars here that look like they wish someone would get in and stomp on the accelerator.
Speaking of accelerators, there's a fun exhibit where you can stand at a little platform and punch the accelerator pedal on the floor, and you'll hear the sound of a specific Porsche zooming off. Naturally, I had to floor it to see how it purred. There's also a big, sit-down video game where you can drive Porsches along the track, and I watched a kid wrack his car against the wall multiple times. Teenagers—no restraint, those creatures.
And if it's more video you're after, the museum has an interactive touchwall that lets you cruise the history of the marque and see a lot of images, history and technical data. You can even compose a musical track that integrates engine sounds (and even things like car door closings) into the mix—and then send the blended track to yourself by email. Too bad that won't include a key.
Don't Forget That Ferdinand Invented the Volkswagen
One of the other intriguing aspects is that the museum dubs itself a "Museum on Wheels"—many of the vehicles are actively driven at events, even the real classics. I'm not sure if they take that 1950 Volkswagen out to race though. There's also a dedicated museum workshop where mechanics regularly service the cars, as well as detail them for your admiration—and you can watch them do it.
The closest I've ever gotten to owning a Porsche is owning four Volkswagens; I suppose if all four of my 1950s and 1960s engines were combined, they might have produced a vehicle that could go 85mph—about what some of the museum cars could do in first gear.
Both museums have restaurants, and I sampled both and approved of both, but that's not good enough—the cars are so delicious looking, you will want to eat them.
[Note: my hosts for these tours were the nice folks at Stuttgart-Marketing GmbH, but all opinions are my own.]
Porsche Sunglasses Rome Museums – Top Museums You Won't Want to Miss Santa Barbara, CA – Museums Exploring the best Art Museums in Barcelona City Guide: Museums in Bloemfontein
Filed Under: Europe · Tagged: Art, Bus Travel, Celebrity, Cruising, Germany, History, Museum, Music, Restaurants, Shopping Mall, Technology, Walking tour
I am one of those people who think of a car as a box with four wheels and you managed to totally capture my attention. I too now want to nibble these cars! Thanks for a great read.
Tom – you need to visit the Marconi Museum in Orange County – talk to Dick Marconi
Tom Bentley says
Allison, cars have been my downfall: I've owned at least 35 of them, many of them vintage creatures that needed a lot of attention that my non-mechanic's hands couldn't supply. So I walked the mechanics over time to my bank carrying bags and said, "Load up." That's why I'm driving a Toyota Corolla (the horror!) today.
Dave, I wasn't even aware of the Marconi Museum. I loved the breadth of the collection—great range of makes and models. I might even be willing to go to Tustin to see it.


Humans are complex animals. For example, think of all that happens in our hearts and minds when we make a decision. The process involves how we think and how we feel, which can change based on recall of memories and reliving past experiences. I remember the rollercoaster ride I took when preparing to ask the girl I loved to marry me—so many imponderables; a lot of questions involving both thinking and feeling. Could I really support her? Was I ready at age twenty three? Could I share the rest of my life with this person? Some questions were analytical in nature, and others were out of pure desire for this person. After spending about twenty minutes in a pensive state, my emotions won! Who cares about all the barriers—I'll figure it out! Can we plan a wedding date? In the end, I made a judgment based on the available information. I'm happy to report that after twenty-eight years of marriage, it was indeed the right choice. I am still madly in love with my wife.
I went to an exhibit on the brain not long ago and was at a loss for words to describe the wonder behind this part of the human anatomy. My attention was drawn to the pituitary gland, a tiny pea-shaped gland at the base of the brain that regulates a number of hormones. Just another facet of human complexity that is so integrated into who we are as people, we often overlook the roles these odd chemicals play in everyday decision-making. We are amazing creatures, and on top of it all, God gives us human personality just to mix it up a bit. We are indeed "fearfully and wonderfully made," as the Psalmist acclaims.
As professional coaches, this admixture of human physiology and personality is what we have to work with when coaching organizational leaders. And, to be effective, we must tap into these same resources—thinking, feeling, rationalizing, learning and judgment to get to the heart of a matter. The reason I prefer the professional coaching platform over other disciplines is because some of my client's decisions are so intense and involve so much risk that the thought of offering my personal opinion seems inappropriate. We know that the best answers come from within our clients, once we have worked with them to ensure all information is available in the decision-making process.
Curiosity is one of the most important ingredients in a coach's mix. I have one client who hired me because he wanted to find another job. So, I'm a coach, right? I'm supposed to follow the client's agenda. This is a slam dunk. He told me his agenda—the client leads with the agenda—the coach follows along and does what the client wants. Right? Wrong!
Being a coach does not mean we shut off our own experiences, judgment, thinking and feeling. We engage all of these facilities in the process of professional coaching. In this particular client relationship we took the time to unpack his world and discover what was behind his thinking and feeling to get to the "who." Rather than taking him and his agenda at face value (which, by the way, happens when we lack empathy), I began to ask him questions. "What are the circumstances that led you to this point of wanting a different job?" "What's behind your desire to change?" His answer revealed that he couldn't get along with his executive vice president and that he had been displaced from being in a managing role, causing him to have bad feelings about his work environment.
After coaching this organizational leader, making hidden information available, he now enjoys working at this same company, reporting to the same executive, and has declared that he is happy in his job. In fact, he is now reaching for more responsibility in the organization and making a real difference. I suspect he will ultimately lead an organization like this some day. Coaching the "who" versus the "what" created a whole shift in mindset for this client.
I mentioned my fetish for the brain. The fact is I love neurology. I worked in Alzheimer's research and also in AIDS research at the University of Pittsburgh in the late 1980s. It was such a cool job. My title was Research Associate and Data Manager, but the best part about the job was having access to research, and some fascinating classes. One of those classes, Brain Cutting, produced feelings of anticipation and enjoyment, much like in elementary school when it was time for recess. I remember it as though it was yesterday—it was my favorite place to go at Pitt.
The professor pulled out a human brain from a jar and proceeded to use a bread knife to slice it into thin pieces. He then passed it around on a cookie sheet for everyone to view. It sounds morbid, but it was so educational and it was inspirational to see the different parts of this human mega-computer. In this particular case, we could see lesions with the naked eye—no microscope needed for this experiment! The learning—AIDS can kill all by itself through degenerative brain disease without resorting to opportunistic infections. What I really learned was how complex and unfathomable this God-given mechanism is, and how we must steward the different types of information we receive and process. So, how does this lesson apply to coaching?
Scientists tell us that there are many different functions of the brain. For all of the emotional intelligence students, we muse over the amygdala, the emotional center of the brain where people process emotions of fear and aggression, for example. What I find interesting is the distinction between the front and the back of the brain; the explicit and implicit learning roles of these two parts. And, while I'm not a scientist (I'm a coach and a scientist wannabe) I resonate with this concept of implicit and explicit learning as it relates to decision-making. I see it in my life. Here's how it works.
When we are faced with a difficult decision we have both thinking and feeling about our choices. Many people refer to implicit learning as "gut feelings." Well, there is some science behind this sense of knowing. It comes from the back of our brain, the experiential learning part of our make-up. Gut feelings are important in decision-making because they often hold data and clues that we just have not taken the time to put into language. For most of us, to make the information available we must think deliberately about our gut feelings and put them into language.
Here's a simple example. Have you ever done something so routine that you didn't even think about it? But then, one day, something feels different and you ask yourself the question, "why?" Think of a woman leaving the mall after a late afternoon shopping venture. She's done this a hundred times. Only this time it feels different. As she's walking into the parking lot, she feels unsafe and decides to call her husband. What just happened? Chances are there was something out of place that told her something was wrong. Perhaps someone she saw out of the corner of her eye seemed suspicious, or reminded her of a feeling she had when she was younger. Who knows—all she knows is that something is out of place, and (hopefully) she takes action.
To return to my earlier example—the executive I mentioned had feelings of relational disconnect and disappointment for not being promoted, or at least given the feedback he thought he deserved to move forward in his career. His first logical, front-of-the-brain type solution was to find another job. By coaching the "who" we were able to center on what was behind his thinking and feeling and do true discovery around his needed action plan. Coaching the "who" created the life changing shift.
With all of this discussion around thinking and feeling, let's talk about turning feelings into facts, the final process before making an important decision. Using my coaching client example, let's look at the process of uncovering his concerns.
Remember that discernment is, by definition, using the right principle at the right time. It is possible to center on a biblical principle that is out of context for the current circumstance and miss the mark.
Using a coach to turn feelings into facts can have a profound impact on one's spiritual growth.
As we learned in our coach training, questions can be our best friends. First, they prevent us from sharing our opinion, if asked properly. Second, they promote thinking in our clients. After all, whose thinking do we like best? The answer is always the same—our own. Leveraging off of our clients thinking is the most profound way to achieve human shifts. As I repeat often, one well placed question is worth more than ten discreet answers. Good questions keep me the coach out of the way, and more transparent in the coaching conversation.
The thinking and feeling aspects of coaching revolve around the explicit and implicit knowledge of our clients. I look at it this way; accessing a client's explicit learning is a venture in unearthing the "what." But, if you want to get to the "who," there is no other option but by asking penetrating questions around life experiences and what's at the heart of one's feelings. This is the place where the who and the what meet, and provide lasting shifts in mindset. Remember that a shift is the basic unit of human change. If we are not facilitating shifts in thinking in our clients, we are probably not really coaching.
This entry was posted on Tuesday, October 6th, 2009 at 9:58 am	and is filed under Articles.


Sally couldn't believe her eyes as she stared at Ross Lorimer. How did he have the nerve to come back? Once, his false promises had left Sally's sister heartbroken.
They had been girls then, but now Sally was a woman. And it was with a woman's feelings that she confronted this so confident man.


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NASA's Lucy Spacecraft Observes Lunar Eclipse From 64 Million Miles Away
TOPICS:AstronomyLucy MissionLunar EclipseNASAPopularSouthwest Research Institute
By Katherine Kretke, Southwest Research Institute May 21, 2022
NASA's Lucy mission, which launched on October 16, 2021, for the first reconnaissance of the Trojans, a population of primitive asteroids orbiting in tandem with Jupiter. In this artist's concept (not to scale), the Lucy spacecraft is flying by Eurybates, one of the six diverse and scientifically important Trojans to be studied. Credit: Southwest Research Institute
NASA's Lucy spacecraft observed the total lunar eclipse on May 15-16, 2022, from a unique vantage point, 64 million miles (100 million km) from the Earth, nearly 70% of the distance between the Earth and the Sun. Using its high-resolution panchromatic camera, L'LORRI, Lucy was able to watch as the Earth cast its shadow on the Moon. At this distance, the Earth and Moon appeared only 0.2 degrees apart to Lucy, having the same separation as a car's tail-lights as viewed from a quarter-mile (400 m) away.
Credit: NASA/Goddard/APL/SwRI
In this timelapse video, the Earth is seen in the left (its rotation clearly visible) while the Moon (on the right, brightened sixfold to increase its visibility) disappears from view as it passes into the Earth's shadow. The video covers a period of almost three hours, from 9:40 p.m. EDT (6:40 p.m. PDT) on May 15 to 12:30 a.m. EDT on May 16 (9:30 p.m. PDT on May 15). The observations ended before the Moon emerged from the shadow.
The Lucy spacecraft launched in October 2021. It is currently traveling back towards Earth for a gravity assist on October 16, 2022, to help propel it on its journey to the Trojan asteroids.
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