Fishing – trapping – and vermin destroying – Fishing – Harpoons and spears
Reexamination Certificate
1994-05-04
2002-03-19
Whisenant, Ethan C. (Department: 1655)
Fishing, trapping, and vermin destroying
Fishing
Harpoons and spears
C536S023100, C536S024300, C536S022100
Reexamination Certificate
active
06357163
ABSTRACT:
The present invention relates to the use of certain nucleic acid analogues in the field of diagnostics, for instance in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities.
Oligodioxyribonucleotides (oligo-DNA's) are finding increasing use in diagnostics procedures. They are for instance finding use in testing for the presence of specific micro-organisms or for testing for the presence of generic predispositions, for instance to disease, in forensic science and in microbiology generally. The uses of oligo-DNA's in this field are of course dependent upon the ability of such oligo-DNA's to hybridise to complementary nucleic acid sequences. By way of example, labelled oligo-DNA probes are used in hybridisation assays to probe immobilised target DNA's for the presence of specific sequences. In amplification procedures, the hybridisation property of oligo-DNA's is utilised to hybridise oligo-DNA primers to template molecules to be amplified.
Oligo-DNA's as long as 100 base pairs in length are now routinely synthesised using a solid support method and fully automatic synthesis machines are commercially available. Attention has nonetheless been given to the possibility of constructing synthetic DNA-analogues capable of hybridising to natural DNA in a sequence specific manner and yet having chemical properties advantageously distinct from DNA itself. This work has been largely motivated by the possible use of such compounds in “anti-sense” therapeutics where the use of conventional oligo-DNA's encounters difficulties because such unmodified oligonucleotides have a short half life in vivo due to the natural presence of nucleases, are difficult and costly to prepare in any quantity and are poor at penetrating cell membranes.
For instance, International (PCT) Patent Application WO86/05518 discloses DNA analogues having a backbone bearing a sequence of ligands, typically nucleotide bases, supposedly capable of sequence specific hybridisation to naturally occurring nucleic acids. A number of different backbone structures are disclosed. No specific exemplification of the provision of such compounds is given and there are no data showing the affinity of the claimed analogues for DNA.
International (PCT) Patent Application WO86/05519 claims diagnostic reagents and systems comprising DNA analogues of the same kind but once again, there is no exemplification.
International (PCT) Patent Application WO89/12060 describes oligonucleotide analogues based on various building blocks from which they are synthesised. Whilst there is exemplification of the building blocks, there is no example of actually preparing an oligonucleotide analogue from them and hence no indication of the performance of the analogues.
Furthermore, it is known to modify the DNA backbone with the aim of increasing resistance to nuclease and generally improving the suitability of the DNA for use in anti-sense therapeutic methods. Other attempts to design DNA analogues are discussed in the introductory portion of WO86/05518 mentioned above.
The universal experience has been that modifications of the backbone of natural DNA lead to a decrease in the stability of the hybrid formed between the modified oligonucleotide and its complementary normal oligonucleotide, assayed by measuring the T
m
value. Consequently, the conventional wisdom in this area is that modifications of the backbone always destabilise the hybrid, i.e. result in lower T
m
values, and therefore the modification should be as minor as possible in order to obtain hybrids with only a slight decrease in T
m
value as the best obtainable result.
The present invention relates to the use in diagnostics or in analysis of nucleic acid analogues of novel structure, preferably having the previously unknown property of forming hybrids with complementary sequence conventional nucleic acids which are more stable in terms of T
m
value than would be a similar hybrid formed by a conventional nucleic acid of corresponding sequence and/or exhibiting greater selectively for the complementary sequence compared to sequences involving a degrees of mis-match than would be exhibited by said corresponding conventional nucleic acid of corresponding sequence.
The invention provides a nucleic acid analogue for use in the capture, recognition, detection, identification or quantitation of one or more chemical or microbiological entities, which analogue is
(a) a peptide nucleic acid (PNA) comprising a polyamide backbone bearing a plurality of ligands at respective spaced locations along said backbone, said ligands being each independently naturally occurring nucleobases, non-naturally occurring nucleobases or nucleobase-binding groups, each said ligand being bound directly or indirectly to a nitrogen atom in said backbone, and said ligand bearing nitrogen atoms mainly being separated from one another in said backbone by from 4 to 8 intervening atoms.
(b) a nucleic acid analogue capable of hybridising to a nucleic acid of complementary sequence to form a hybrid which is more stable against denaturation by heat than a hybrid between the conventional deoxyribonucleotide corresponding to said analogue and said nucleic acid; or
(c) a nucleic acid analogue capable of hybridising to a double stranded nucleic acid in which one strand has a sequence complementary to said analogue, so as to displace the other strand from said one strand.
The separation of the nitrogen bearing atoms in the backbone of nucleic acid analogues defined in paragraph (a) above (PNA's) is preferably by five atoms. In nucleic acid analogues having the Formula I (below) this has been found to provide the strongest affinity for DNA. However, it may in some cases be desired to reduce the strength of binding between the PNA's and DNA by spacing one or more of the ligands by a less than optimal spacing, e.g. by a spacing of more than 5 atoms, e.g. by up to 14 atoms or more.
Preferably not more than 25% of interligand spacings will be 6 atoms or more. More preferably not more than 10 to 15% of interligand spacings will be 6 atoms or more. The aza nitrogen atoms which carry the ligands (directly or via linker groups are not themselves counted in the spacings referred to above.
An alternative or additional method for reducing the strength of DNA binding is to omit certain of the ligands, putting in their place a moiety which contributes little or nothing to the binding of DNA, e.g. hydrogen. Preferably, not more than 25% of the ligand positions will be occupied by non-binding moieties, e.g. not more than 10 to 15%.
Representative ligands include either the four main naturally occurring DNA bases (i.e., thymine, cytosine, adenine or guanine) or other naturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosine or thiouracil) or artificial bases (e.g., bromouracil, azaadenines or azaguanines, etc.) attached to a peptide backbone through a suitable linker.
In preferred embodiments, the nucleic acid analogues used in the invention have the general formula (I):
wherein:
n is at least 2,
each of L
1
-L
n
is independently selected from the group consisting of hydrogen, hydroxy, (C
1
-C
4
)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups and reporter ligands, at least one of L
1
-L
n
being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
each of A
1
-A
n
is a single bond, a methylene group or a group of formula (IIa) or (IIb):
where:
X is O, S, Se, NR
3
, CH
2
or C(CH
3
)
2
;
Y is a single bond, O, S or NR
4
;
each of p and q is an integer from 1 to 5, the sum p+q being not more than 10;
each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10;
each R
1
and R
2
is independently selected from the group consisting of hydrogen, (C
1
-C
4
)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alk
Berg Rolf H.
Buchardt Ole
Egholm Michael
Nielsen Peter E.
Pillsbury & Winthrop LLP
Whisenant Ethan C.
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