Nucleic acid analogs with a chelating functionality

Details Nucleic acid analogs with a chelating functionality Nucleic acid analogs with a chelating functionality

1998-05-29

2002-02-12

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S501000, C530S300000, C530S350000, C536S023100

Reexamination Certificate

active

06346378

ABSTRACT:

The present invention relates to nucleic acid analogs having a chelation functionality, to their uses in assay procedures, to methods of capturing them to solid supports and to methods of concentrating solutions of them.
Nucleic acid analogs having important new utilities in assay procedures and in the field of diagnostics have been described in WO 92/20703. These nucleic acid analogs had a number of new properties making them of special importance in the field of diagnostics as well as in the field of antisense therapeutics.
They typically feature a polyamide backbone bearing a sequence of ligands which are nucleic acid bases. The analogs described there have the property of hybridizing with great specificity and stability to natural nucleic acids of complementary sequence.
In order to aid the detection and the manipulation of such nucleic acid analogs in diagnostics or other assay procedures and the like operations, it is desirable to provide the nucleic acid analogs with detectable labels. It is also desirable to find ways of capturing said nucleic acid analogs on solid supports. Various labels are described in WO 92/20703. Also, the capture of the nucleic acid analogs to solid supports via bound nucleic acid or nucleic acid analog sequences acting as capture probes is described.
However, it is desirable to find alternative capture methods and in particular methods which do not require a tailored capture probe which is sequence specific but rather are generally applicable to such nucleic acid analogs.
In EP-A-0 097 373 the synthesis of nucleic acids labeled with a complexing agent is described. However, the synthesis of these compounds appears to be complicated.
Furthermore, whilst natural nucleic acids are readily and routinely concentrated by precipitation from solution by ethanol, centrifugation and resuspension, no such convenient method presently exists to aid those working with these nucleic acid analogs.
The present invention now provides according to a first aspect thereof a nucleic acid analog comprising a polymeric strand which includes a sequence of ligands bound to a backbone made up of linked backbone moieties, which analog is capable of hybridization to a nucleic acid of complementary sequence, further comprising, preferably at one terminus of said backbone a chelating moiety capable of binding at least one metal ion by chelation.
Preferably, the backbone is a polyamide, polythioamide, polysulphinamide or polysulphonamide backbone and preferably said chelating moiety is present at the N-terminus.
The chelating moiety preferably comprises a sequence of peptide bonded amino acids.
Preferred sequences of amino acids for use as chelating moieties are -His, Gly,Asp or -(His)
n
, where n=3 to 10, e.g. 5 or 6. The longer sequences may bind more than one metal ion per molecule of nucleic acid analog.
Alternatively, said chelating moiety may be a polycarboxylic acid substituted amine such as ethylenediamine-tetraacetic acid (EDTA) or aminotriacetic acid (NtA) and the like.
The nucleic acid analog is preferably capable of hybridizing 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 in sequence to said analog and said nucleic acid.
Said nucleic acid analog is preferably a peptide nucleic acid in which said backbone is a polyamide backbone, each said ligand being bonded 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.
The analog is preferably capable of hybridizing to a double stranded nucleic acid in which one strand has a sequence complementary to said analog, in such a way as to displace the other strand from said one strand.
More preferred PNA compounds for use in the invention have the formula:
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, heterocyclic moieties, reporter ligands and chelating moieties;
each of C
1
-C
n
is (CR
6
R
7
)
y
(preferably CR
6
R
7
, CHR
6
CHR
7
or CR
6
R
7
CH
2
) where R
6
is hydrogen and R
7
is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R
6
and R
7
are independently selected from the group consisting of hydrogen, (C
2
-C
6
)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C
1
-C
6
)alkoxy, (C
1
-C
6
)aIkylthio, NR
3
R
4
and SR
5
, where R
3
and R
4
are as described below, and R
5
is hydrogen, (C
1
-C
6
)alkyl, hydroxy, alkoxy, or alkylthio-substituted (C
1
to C
6
)alkyl or R
6
and R
7
taken together complete an alicyclic or heterocyclic system;
each of D
1
-D
n
is (CR
6
R
7
)
z
(preferably CR
6
R
7
, CHR
6
CHR
7
or CH
2
CR
6
R
7
) where R
6
and R
7
are as defined above;
each of y and z is zero or an integer from 1 to 10, the sum y+Z being at least 2, preferably greater than 2, but not more than 10;
each of G
1
-G
n-1
is —NR
3
CO—, —NR
3
C
5
—, —NR
3
SO— or —NR
3
SO
2
—, in other orientation, where R
3
is as defined below;
each of A
1
-A
n
and B
1
-B
n
are selected such that:
(a) A is a group of formula (lla), (llb), (llc) or (lld), and B is N or R
3
N
+
; or
(b) A is a group of formula (lld) and B is CH;
wherein:
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 zero or 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, alkoxy, alkylthio, amino and halogen; and
each R
3
and R
4
is independently selected from the group consisting of hydrogen, (C
1
-C
4
)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C
1
-C
4
)alkyl, hydroxy, alkoxy, alkylthio and amino;
Q is —CO
2
H, —CONR′R″, —SO
3
H or —SO
2
—NR′R″ or an activated derivative of —CO
2
H or —SO
3
H; and
I is —NR′R″′ where R′ and R″ are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, nucleosides, nucleotides, nucleotide diphosphates, nucleotide triphosphates, oligonucleotides, including both oligoribonucleotides and oligodeoxyribonucleotides, oligonucleosides and soluble and non-soluble polymers, and —R″′ is a chelating moiety. “Oligonucleosides” includes nucleobases bonded to ribose and connected via a backbone other than the normal phosphate backbone of nucleic acids.
In the above structures wherein R′ or R″ is an oligonucleotide or oligonucleoside, such structures can be considered chimeric structures between PNA compounds and the oligonucleotide or oligonucleoside.
Generally, at least one of L
1
-L
n
will be a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase binding group.
Preferred PNA-containing compounds useful to effect binding to RNA, ssDNA and dsDNA and to form triplexing structures are compounds of the formula III, IV or V:
wherein:
each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;
each R
7
is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;
n is an integer greater than 1,
each k, l, and m is, independently, zero or an integer from 1 to 5;
each p is zero or 1;
R
h
is OH, NH
2
or —NHLysNH
2
; and
R′ is a chelating moiety.
The invention includes according to a second aspect thereof a method of capturi

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