Labeling of oligonucleotide probes by multiple-way ligation
View Patent ↗The present invention provides a novel method for multiple labelling of nucleic acid probes by ligation. The method uses a ligase catalysed reaction to connect a nucleic acid probe with multiple pre-prepared nucleic acid label carrier molecules under the presence of a stabilizing complementary adaptor oligonucleotide. The method allows for an easy, cheap and fast labelling of multiple probes with multiple different labels. In this way, the costs and effort for the generation of single molecule Fluorescent In Situ Hybridization (smFISH) assays was significantly reduced, allowing combinatorial multi-color barcoding of nucleic acid probes The invention further provides methods for the generation of FISH libraries and labelling kits comprising the novel tools of the invention.
1 . A method for producing a labeled, or otherwise modified, oligonucleotide probe, the method comprising the steps of:
(a) providing a probe-sequence-oligonucleotide comprising (i) a probe sequence comprising a nucleotide sequence complementary to a target nucleic acid and (ii) a predetermined tag nucleotide sequence complementary to a first adaptor nucleotide sequence,
(b) providing a label-carrier-oligonucleotide comprising at least one labeling moiety, or other functional moiety, wherein the label-carrier-oligonucleotide has a predetermined tag nucleotide sequence complementary to a second adaptor nucleotide sequence,
(c) providing an adaptor-oligonucleotide, comprising in direct sequence and on a single strand, the first and the second adaptor nucleotide sequence;
(d) bringing into contact under hybridizing conditions the probe-sequence-oligonucleotide, the label-carrier-oligonucleotide and the adaptor-oligonucleotide, to form a complex, wherein, in the complex, a free/unblocked 3′-OH group is in close spatial proximity to a free/unblocked 5′ phosphate group,
(e) reacting the complex to form a covalent bond between the 3′-OH group and the 5′-phosphate group using a ligase under ligating conditions, and
(f) removing the adaptor-oligonucleotide to form the labeled, or otherwise modified, oligonucleotide probe.
2 . The method for producing the labeled, or otherwise modified, oligonucleotide probe according to claim 1 , wherein:
in the probe-sequence-oligonucleotide, the probe sequence is 5′ of the predetermined tag nucleotide sequence,
in the adaptor-oligonucleotide, the first and second adaptor nucleotide sequence are in 3′ to 5′ direction in direct sequence,
the free/unblocked 3′-OH group is located on the probe-sequence-oligonucleotide and the free/unblocked 5′-phosphate group is located on the label-carrier-oligonucleotide, and
in the complex in step (d), the free/unblocked 3′-OH group of the probe-sequence-oligonucleotide is in close spatial proximity to the free/unblocked 5′-phosphate group of the label-carrier-oligonucleotide.
3 . The method for producing the labeled, or otherwise modified, oligonucleotide probe according to claim 1 , wherein:
in the probe-sequence-oligonucleotide, the probe sequence is 3′ of the predetermined tag nucleotide sequence,
in the adaptor-oligonucleotide, the first and second adaptor nucleotide sequence are in 5′ to 3′ direction in direct sequence,
the free/unblocked 3′-OH group is located on the label-carrier-oligonucleotide and the free/unblocked 5′-phosphate group is located on the probe-sequence-oligonucleotide, and
in the complex in step (d), the free/unblocked 5′-phosphate group of the probe-sequence-oligonucleotide is in close spatial proximity to the free/unblocked 3′-OH group of the label-carrier-oligonucleotide.
4 . The method for producing the labeled, or otherwise modified, oligonucleotide probe according to claim 1 , wherein step (b) comprises providing a first, and a second, optionally a third or more, label-carrier-oligonucleotides, wherein the first label-carrier-oligonucleotide has a predetermined tag nucleotide sequence complementary to a second adaptor nucleotide sequence, the second label-carrier-oligonucleotide has a predetermined tag nucleotide sequence complementary to a third adaptor nucleotide sequence, optionally, the third or more label-carrier-oligonucleotide has a predetermined tag nucleotide sequence complementary to a fourth or more adaptor nucleotide sequence, and wherein in step (c) the adaptor-oligonucleotide comprises in direct sequence the first, the second, the third, optionally the fourth or more, adaptor nucleotide sequence.
5 . The method according to claim 4 , wherein each of the adaptor nucleotide sequences comprises distinct nucleotide sequences.
6 . The method according to claim 4 , wherein each of the label-carrier-oligonucleotides is differently labeled or otherwise modified.
7 . The method according to claim 1 , wherein the label-carrier-oligonucleotide comprises two or more labeling moieties.
8 . The method according to claim 7 , wherein the two or more, labeling moieties comprise at least two or more different labeling moieties, or other functional moieties.
9 . The method according to claim 1 , comprising purifying the labeled, or otherwise modified, oligonucleotide probe.
10 . The method according to claim 1 , wherein the probe-sequence-oligonucleotide has a length of between 20 to 300 nucleotides.
11 . A method for generating a single molecule Fluorescent In-Situ Hybridization (smFISH) probe library, comprising producing at least two fluorescent labeled oligonucleotide probes according to the method of claim 1 , wherein the labeling moiety is a fluorescent moiety, and wherein the at least two fluorescent oligonucleotide probes are capable of binding to one target nucleic acid.
12 . The method according to claim 11 , wherein the at least two fluorescent labeled oligonucleotide probes are at least 10 fluorescent labeled oligonucleotide probes, and wherein each of the fluorescent labeled oligonucleotide probes is capable of binding to the one target nucleic acid.
13 . The method according to claim 1 , wherein the labeled, or otherwise modified, oligonucleotide probe is single-stranded.