5′-modified nucleotides and the application thereof in...

Details 5′-modified nucleotides and the application thereof in... 5′-modified nucleotides and the application thereof in...

2001-01-10

2003-09-30

Riley, Jezia (Department: 1637)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200, C435S128000, C435S130000, C536S022100, C536S023100, C536S025300

Reexamination Certificate

active

06627416

ABSTRACT:

The invention relates to 5′-modified nucleotides and to nucleic acids which contain these nucleotides. Processes for incorporating the 5′-modified nucleotides into nucleic acids, and the subsequent site-specific cleavage of the nucleic acids at the 5′-modified monomer building blocks, are also disclosed. These processes can be employed for nucleic acid sequencing, for generating nucleic acid libraries, for detecting mutations, for preparing support-bound nucleic acids and for pharmaceutical purposes.
The processes which are nowadays routinely used for sequencing nucleic acids generally comprise polymerizing a nucleic acid strand which is complementary to a template and generating a mixture of nucleic acid fragments of all possible lengths (1). This nucleic acid fragment mixture can be obtained by terminating the polymerization or degrading using exonucleases (2), by iterative sequencing methods (3), by adding individual bases and detecting the release of pyrophosphate (4), by chemical methods using elimination reactions (5), by chemicoenzymic methods, involving incorporating modified nucleosides and cleaving by attack on phosphorothioate- or boron-modified nucleotides (6), by incorporating ribonucleosides into DNA and subsequently cleaving under basic conditions (7) or by incorporating 3′-dye-labelled nucleotides while at the same time or subsequently eliminating the dye (8). In addition to these methods, strategies are also available which involve sequencing by hybridizing (9) and a physical production of fragments by means of mass spectrometry (10). The possibility of detecting by means of atomic force microscopy (11) has also been discussed.
However, in the last twenty years, the method of choice has been the enzymic chain termination method. This method makes possible automation and sequencing with high throughput for use when sequencing entire genomes. The automation was achieved by using dye primers (12), internal labelling (13) or dye terminators (14). Sequencing with dye primers and internal labelling suffer, however, from the disadvantage that irregular termination events occur in the sequence ladder and can lead to erroneous interpretation of the sequence data. Dye terminators suffer from the disadvantage that they are sometimes incorporated at incorrect sites and only permit a limited length to be read since they are modified substrates.
In this addition to this, there is a need to reduce the quantity of DNA which is required for a sequence determination. There is currently only one single cyclic sequencing method available for this purpose (15), which method, however, in contrast to PCR, in which an exponential amplification takes place, only leads to linear amplification of the products. The direct sequencing of PCR products in turn displays disadvantages since relatively large quantities of triphosphates and primer molecules are present in the reaction vessels and can lead to impairment of the sequencing reaction or the sequence determination (16). However, the purification of the PCR products is time-consuming and represents an additional procedural step. While triphosphates can be cleaved using enzymic methods (17), this is also time-consuming and increases the costs of carrying out the sequencing reaction. As an alternative, a direct exponential amplification and sequencing method (DEXAS) is available for sequencing small quantities of DNA material (18); however, it has so far not been possible to use this method for a standard sequencing and, in contrast to its name, the method is not directly exponential.
The present invention makes available a novel process for sequencing nucleic acid, which process at least partially avoids the disadvantages of the state of the art. In particular, this process avoids the problem of substrate specificity with regard to dye terminators and makes possible rapid DNA sequencing using very small quantities of DNA starting material in combination with a nucleic acid amplification reaction such as PCR. The process also improves the readable length of the sequenceable templates.
The process according to the invention is based on using compounds of the general formula (I):
in which:
B denotes a nucleobase, i.e. a natural or unnatural base which is suitable for hybridizing to complementary nucleic acid strands, such as A, C, G, T, U, I, 7-deaza-G, 7-deaza-A, 5-methyl-C, etc.,
W and Z in each case denote OR
1
, SR
1
, N(R
1
)
2
or R
1
, where R
1
, in each case independently, on each occurrence represents hydrogen or an organic radical, e.g. an alkyl, alkenyl, hydroxyalkyl, amine, ester, acetal or thioester radical, preferably having up to 10 carbon atoms and particularly preferably having up to 6 carbon atoms,
X denotes OR
2
, SR
2
or B(R
2
)
3
, where R
2
, in each case independently, denotes hydrogen, a cation, e.g. an alkali metal ion or ammonium ion, or an organic radical, e.g. a dye such as fluorescein, rhodamine, cyanine and their derivatives,
Y denotes NR
3
or S, in particular NR
3
, where R
3
represents hydrogen or an organic radical, e.g. a saturated or unsaturated hydrocarbon radical, in particular a C
1
-C
4
radical or a dye radical, with hydrogen also being understood to mean the isotopes deuterium and tritium, and
R denotes hydrogen, a cation, an organic radical or an optionally modified phosphate group or diphosphate group, in particular a diphosphate group,
for incorporation into nucleic acids and for the subsequent site-specific cleavage of the nucleic acids, preferably by hydrolysing the P—Y bond, resulting in the formation of nucleic acid fragments having an HY—CH
2
-5′ end.
The group R can denote an organic radical, for example a lipophilic radical, which facilitates the penetration of the substance into a cell. R is preferably a phosphate group:
or a diphosphate group:
This phosphate or diphosphate group can be modified. Thus, one or more terminal oxygen atoms can carry substituents, e.g. organic radicals. On the other hand, one or more terminal oxygen atoms and, in the case of the diphosphate group, the bridging oxygen atom as well, can be replaced by groups such as S, NR
3
or C(R
3
)
2
, with R
3
being defined as before. In addition to this, 2 substituents on terminal oxygen atoms can also be bridged with each other.
When substituents are present, they are preferably located on oxygen atoms belonging to the phosphorus atom which is in each case terminal, particularly preferably on the &ggr;-phosphorus atom. Examples of suitable substituents are organic radicals such as alkyl radicals, which can themselves be substituted, or a salicyl group, which can form a 6-membered cyclic diester with 2 oxygen atoms belonging to the terminal phosphorus. The aromatic nucleus of the salicyl groups can again itself carry one or more additional substituents, e.g. those defined as for R
1
or halogen atoms. Additionally preferred substituents on the oxygen atom are radicals such as C
1
-C
10
-alkyl, —(CH
2
)
n
—N
3
, (CH
2
)
n
N(R
3
)
2
or —(CH
2
)
n
NHOCO(CH
2
)
m
—N(R
3
)
2
, where n and m are integers from 1 to 8, preferably from 2 to 5, and R
3
is defined as above, but can, in addition, preferably denote an aromatic radical such as phenyl or dinitrophenyl.
The incorporation of compounds of the general formula (I) into nucleic acids preferably takes place enzymically. However, a chemical synthesis is also possible. For an enzymic incorporation, preference is given to using enzymes which are selected from the group consisting of DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, RNA-dependent RNA polymerases and terminal transferases. Particular preference is given to T7 DNA polymerase and related enzymes, such as T3 DNA polymerase or SP6 DNA polymerase, or modifications of these enzymes. Correspondingly, the nucleic acids into which the compounds of the formula (I) are incorporated can be DNAs and/or RNAs which can, where appropriate, carry one or more additional modified nucleotide building blocks.
Nucleic acids which contain, as monomeric bui

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