Template Assembly by Proximity-Enhanced Reactivity via Metabolic Labeling
OwnerJames T. Kurnick, Ian Seymour Dunn +1inventors
InventorsJames T. Kurnick, Ian Seymour Dunn, Matthew M. Lawler
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Abstract
The present disclosure provides methods of metabolically labeling cells to provide a substrate for assembly of desired molecules, and to bifunctional compounds used in such methods.
Claims173
A method of labeling a cell with a substrate for a templated assembly reaction, the method comprising the steps:
- a) contacting the cell with an azide-modified sugar;
- and
- b) contacting the cell with the substrate for the templated assembly reaction, wherein the substrate comprises:
- i) a nucleic acid template;
- and
- ii) an azide reactive molecule linked to the nucleic acid template at the 5′- or 3′-end of the nucleic acid template, wherein the azide reactive molecule is chemically reactable with the azide of the azide-modified sugar.
A method of labeling a cell surface with a quantifiable reverse template, the method comprising the steps:
- a) contacting the cell with an azide-modified sugar;
- b) contacting the cell with two ligation-template oligonucleotides, wherein:
- i) the first ligation-template oligonucleotide (LT1) comprises a 5′-azide reactive molecule that is chemically reactable with an azide group;
- and
- ii) the second ligation-template oligonucleotide (LT2) comprises a 5′-phosphate, and a 3′-azide reactive molecule that is chemically reactable with an azide group;
- c) contacting the cell with a nuclease resistant oligonucleotide, wherein the nuclease resistant oligonucleotide is non-overlap complementary to both LT1 and LT2;
- wherein upon close proximity, the 3′—OH of LT1 and the 5′-phosphate of LT2 are ligatable;
- and
- d) contacting the cell with a ligase, thereby generating a reverse template formed from LT1 and LT2 that can be amplified and quantified.
A method of labeling a cell with a substrate for a templated assembly reaction, the method comprising the steps:
- a) contacting the cell with an azide-modified sugar;
- and
- b) contacting the cell with a nucleic acid template, wherein the nucleic acid template comprises:
- i) a first hybridization region and a second hybridization region separated by a loop region, wherein the first hybridization region is complementary to the second hybridization region;
- and
- ii) a azide reactive molecule at the 3′-end of the nucleic acid template and at the 5′-end of the nucleic acid template, wherein both azide reactive molecules are chemically reactable with an azide group.
A method of metabolically labeling the surface of a specific target cell, the method comprising the steps:
- a) contacting the cell with a nucleic acid molecule, wherein the nucleic acid molecule comprises:
- i) a short terminal RNA segment comprising a terminal azide-modified sugar;
- and
- ii) a longer modified RNA segment linked to the short terminal RNA segment, wherein the terminal end of the longer modified RNA segment is complementary to the short terminal RNA segment;
- wherein the longer modified RNA segment is complementary to a specific transcript target within the specific targeted cell;
- and
- wherein the longer modified RNA segment is modified to be nuclease resistant.
A method of metabolically labeling the surface of a cell, the method comprising the steps:
- a) contacting the cell with a nucleic acid molecule, wherein the nucleic acid molecule comprises:
- i) a short terminal RNA segment comprising a terminal azide-modified sugar;
- ii) a longer modified RNA segment, wherein a first terminal end of the longer modified RNA segment is complementary to the short terminal RNA segment;
- and
- iii) a short linker nucleic acid having a first terminal end linked to the terminal end of the short terminal RNA segment that does not comprise the azide-modified sugar wherein the short linker nucleic acid is complementary to a second terminal end of the longer modified RNA segment that is not complementary to the short terminal RNA segment;
- wherein the longer modified RNA segment is complementary to a specific transcript target;
- and
- wherein the longer modified RNA segment is modified to be nuclease resistant.
Dependencies are inferred from the claim text.