Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-12-17
2002-04-23
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S025300, C536S025310, C536S025320, C540S465000, C540S474000, C556S001000, C556S006000
Reexamination Certificate
active
06376179
ABSTRACT:
The present invention relates to a novel process for labelling a synthetic or natural ribonucleic acid (RNA).
A synthetic RNA is to be understood as meaning an RNA which is obtained by a technique which was developed by man, for example an amplification technique (PCR followed by a transcription) or a transcriptional amplification technique (TMA). A natural RNA is to be understood as meaning an RNA which is obtained by extraction from a cell, for example a messenger RNA, a ribosomal RNA or a transfer RNA.
The state of the art shows that there are a large number of methods for labelling nucleotides, oligonucleotides or nucleic acids; oligonucleotides and nucleic acids will be referred to by the term polynucleotides. Oligonucleotides can be labelled either during synthesis or by incorporating at least one labelled nucleotide.
A first method consists in attaching the label to the base, whether the latter is a natural base or a modified base. A second method proposes attaching the label to the sugar, again whether the latter be a natural sugar or a modified sugar. A third method relates to attaching the label to the phosphate.
Labelling the base was used, in particular, in the approach of labelling nucleic acids by incorporating directly labelled nucleotides.
Labelling the sugar is often used in the case of nucleic acid probes which are prepared by chemical synthesis.
Labelling the phosphate has also been used for introducing functionlized arms and labels when synthesizing the polynucleotides chemically.
In fact, the skilled person who is to label a nucleotide or a nucleotide analogue or a nucleic acid is inclined to attach the label to the base or to the sugar, which offer him more convenience and more options. This is, furthermore, what emerges from studying a large number of documents such as EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879, U.S. Pat. No. 5,449,767, U.S. Pat. No. 5,328,824, WO-A-93/16094, DE-A-3.910.151 and EP-A-0.567.841 in the case of the base or EP-A-0.286.898 in the case of the sugar.
Nevertheless, these techniques suffer from a number of drawbacks, the two main ones being the steric hindrance and the effects which are engendered by the presence of a label.
When the base is labelled, the steric hindrance is due to the encroachment of the label on the space where a neighbouring base is present, irrespective of whether this neighbouring base is carried by an adjacent nucleotide of the same strand or by the complementary strand. It is also quite obvious that the presence of the label on the base can impair efficacy and specificity during enzymic incorporation and can have an effect on the quality of the hydrogen bonds between the two complementary strands, something which can be injurious to hybridization.
When the sugar is labelled, the steric hindrance is due to the encroachment of the label on the space where an adjacent sugar carried by the same strand is present. This presence of the label can move apart two adjacent bases which are carried by the same strand and, as a consequence, prevent satisfactory hybridization with the complementary strand due to the fact that the hydrogen bonds between the strands are not optimal.
The technique of attaching the label to the phosphate is more complex than the technique involved in functionalizing the base or the sugar.
Even so, some documents have proposed techniques for labelling the phosphate. This applies, for example, to document EP-A-0.280.058, which describes labelling a nucleotide by attaching the label to the phosphate, with the latter being attached to the sugar in the 2′ and/or 5′ positions, when the nucleotide is a deoxyribonucleotide, and in the 2′, 3′ and/or 5′ positions when the nucleotide is a ribonucleotide. This document also describes a polynucleotide or oligonucleotide which comprises at least one labelled nucleotide as described above; this nucleotide is incorporated into the polynucleotide or oligonucleotide during synthesis.
However, the labelling which is proposed by document EP-A-0.280.058 does not enable the nucleic acids to be labelled uniformly. This is because the incorporation of the labelled nucleotides into the polynucleotides cannot be controlled; it depends entirely on the composition of polynucleotides which is to be synthesized. Thus, some polynucleotides may contain a large number of labelled nucleotides whereas others may not contain any at all. As a result, the intensity of the signal emitted by these nucleic acids will not be uniform, something which could easily make it difficult to interpret the results when detecting the nucleic acids.
In this case, the labelling is incorporated biologically without there being any control of the positions of the labelled nucleotides.
The document U.S. Pat. No. 5,317,098 relates to nucleic acids which are labelled at their 5′ ends. This attachment uses imidazole and a linker arm. There is no associated fragmentation. Furthermore, phosphate is added; kinase is therefore used.
Nevertheless, a phosphate will logically be present at each free end of the nucleic acid, leading to at least one additional step. This labelling is not associated with any fragmentation.
In addition, the labelling described by the preceding two documents is carried out on large nucleic acids. Thus, no fragmentation stage, also termed a cleavage stage, wets described before the labelling steps. As a result, the duplexes formed after hybridization are not stable when these target nucleic acids are hybridized to capture probes. This also applies when the polynucleotides are used as detection probes. The reasons may be due to steric hindrance or to a lack of specificity between the polynucleotide, which has been synthesized, and its target, which is not necessarily of the same size. This will therefore result in a quantitative and qualitative loss of the signal.
Steric hindrance may not only be the result of the length of the nucleic acid but also of the existence or the conservation of secondary structures. Fragmentation makes it possible to destroy these structures and in this way to optimize hybridization. This steric hindrance plays a particularly important role in the case of hybridization to surfaces which contain a high density of capture probes, for example the DNA chips developed by the company Affymetrix (“Accessing Genetic Information with High-Density DNA arrays”, M. Shee et al., Science, 274, 610-614. “Light-generated oligonucleotide arrays for rapid DNA sequence analysis”, A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 5022-5026). In this technology, the capture probes are generally of reduced size, being of about twenty nucleotides.
A large number of methods are described in the state of the art for fragmenting nucleic acids.
In the first place, the fragmentation can be enzymic, i.e. the nucleic acids can be fragmented by nucleases (DNases or RNases). This generates small fragments having 3′-OH, 5′-OH, 3′-phosphate and 5′-phosphate ends.
In the second place, the fragmentation can be chemical. For example, in the case of DNAs, it is possible to depurinate or depyrimidinate the DNAs, which are then fragmented in the presence of a base by a mechanism termed “&bgr;-elimination”. The DNAs can be fragmented by oxidation, alkylation or free radical addition mechanisms, inter alia. Metal cations, which are often combined with organic molecules used as chemical catalysts, for example imidazole, are used for fragmenting RNAs. This fragmentation is preferably carried out in an alkaline medium and generates fragments having 3′-phosphate ends.
However, the objective of these fragmentations is not that of facilitating or permitting labelling.
Document WO-A-88/04300 proposes a method for fragmenting and labelling RNA, with the fragmentation being carried out using RNA which possesses enzymic properties, i.e. ribozymes. With each cleavage, this fragmentation by ribozymes releases a nucleic acid (5′) HO end and a nucleic acid (3′) HO—PO
2
end. The labelling, which is
Bio Merieux
Chakrabarti Arun Kr.
Fredman Jeffrey
Oliff & Berridg,e PLC
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