Double stranded nucleic acid biochips
This invention describes a new method of constructing double-stranded DNA (dsDNA) microarrays based on the use of pre-synthesized or natural DNA duplexes without a stem-loop structure. The complementary oligonucleotide chains are bonded together by a novel connector that includes a linker for immobilization on a matrix. A non-enzymatic method for synthesizing double-stranded nucleic acids with this novel connector enables the construction of inexpensive and robust dsDNA/dsRNA microarrays. DNA-DNA and DNA-protein interactions are investigated using the microarrays.
1. A nucleotide-matrix connector comprising a bifunctional phosphoramidite and a nitrogen linker atom adapted for connecting to the matrix, the connector having the formula:
wherein DMTr is dimethoxytrityl and the connector, as part of a nucleic acid strand enables the formation of a synthetic duplex structure and immobilizes the synthetic duplex on the matrix.
2. A nucleic acid comprising strands linked by the connector of claim 1 .
3. The nucleic acid of claim 2 is DNA.
4. The nucleic acid of claim 2 is RNA.
5. The nucleic acid of claim 2 is a DNA-RNA hybrid.
6. The nucleic acid of claim 2 is a full duplex.
7. The nucleic acid of claim 2 is a partial duplex with a 3′-protruding end.
8. The nucleic acid of claim 2 is a partial duplex with a 5′-protruding end.
9. The nucleic acid of claim 2 is synthetic.
10. A method for synthesizing a connector designated phosphoramidite of claim 1 , said method comprising:
(a) synthesizing an activated ester of aminocaproic acid by
(i) protecting the NH 2 group of 6-aminocaproic acid with trifluoraccetic protective group; and
(ii) reacting with N-hydroxysuccinimide and N,N-dicylohexylcarbodiimide;
(b) obtaining a serinol derivative through
(i) reacting sequentially with ethyl trifluroacetate and 4,4′-dimethoxytritylchloride and
(ii) treating with methylamine;
(c) condensing the serinol derivative with an activated ester of N-trifluoroacetyl-6-aminocaproic acid; and
(d) phosphitylating the obtained compound with 2-cyanoethyl diisopropylchlorophosphoramidite.
11. A method for constructing double-stranded nucleic acid biochips (microarrays), said method comprising:
(a) synthesizing at least one double-stranded nucleic acid in which complementary oligonucleotide chains are attached together by the connector of claim 1 ; and
(b) attaching the synthesized double-stranded nucleic acid to a matrix through a free-amino group of the linker.
12. A method for constructing a double-stranded nucleic acid microarray, said method comprising:
(a) synthesizing at least one double-stranded nucleic acid with the connector of claim 1 with at least one specific sequence for restriction endonuclease recognition;
(b) attaching the synthesized double-stranded nucleic acid to a matrix;
(c) digesting the attached double-stranded nucleic acid on the matrix using appropriate restriction endonucleases; and
(d) annealing the digested double-stranded nucleic acid on the matrix with double-stranded nucleic acid fragments having complementary protruding ends; and
(e) ligating the annealed result of (d).
13. The method of claim 12 , wherein the double-stranded nucleic acid is DNA.
14. The method of claim 12 , wherein the double-stranded nucleic acid is RNA.
15. The method of claim 12 , wherein the double-stranded nucleic acid is a DNA-RNA hybrid.
16. A method for investigating double-stranded nucleic acid-protein interactions, said method comprising:
(a) synthesizing a double-stranded nucleic acid probe of a specific sequence having a connector as in claim 1 ;
(b) attaching the synthesized double-stranded nucleic acid probe to a matrix;
(c) providing conditions necessary for the double-stranded nucleic acid-protein interaction; and
(d) determining results of the interaction.
17. The method of claim 16 , wherein the double-stranded nucleic acid is DNA.
18. A method for non-enzymatically synthesizing a double-stranded nucleic acid without forming a stem-loop structure, said method comprising:
synthesizing a linear chain of complementary nucleic acids with the connector of claim 1 at a specific position within the chain; and
providing conditions necessary for double-strand structure formation from the complementary chains.
19. A method to construct double-stranded DNA microarrays, said method comprising:
(a) synthesizing complementary oligonucleotide chains bonded together by a novel connector to form a DNA duplex;
(b) incorporating a linker to the connector, said linker capable of anchoring the DNA duplex to a support; and
(c) contacting the chemically synthesized duplex-connector-linker molecule to a support.
20. A microchip with dsDNA oligonucleotides each anchored to gel pads by the connector of claim 1 .
21. A dsDNA microarray constructed using the method of claim 12 .