OLIGONUCLEOTIDES AND METHODS FOR PREPARING

Abstract

Oligonucleotide constructs are described, each comprising a functional element and a coding element, wherein the functional element comprises a functional sequence, the functional sequence comprising a sequence of nucleotides in which one or more, or each, nucleotide is modified and the coding element comprises a coding sequence, the coding sequence comprising a sequence of nucleotides which do not contain the modifications of the functional sequence, wherein the coding sequence encodes the sequence structure of the functional sequence.

Claims

1-38. (canceled)

39-76. (canceled)

77. An oligonucleotide construct comprising a functional element and a coding element, wherein the functional element comprises a functional sequence, the functional sequence comprising a sequence of nucleotides in which one or more, or each, nucleotide is modified and the coding element comprises a coding sequence, the coding sequence comprising a sequence of nucleotides which do not contain the modifications of the functional sequence, wherein the coding sequence encodes the sequence structure of the functional sequence and wherein the coding sequence is an oligonucleotide polymer of n-mers, or a plurality of different n-mers, where n is an integer of 2 or greater and each n-mer corresponds to a single nucleotide in the functional sequence and the coding sequence has n times the number of nucleotides as the functional sequence.

78. The oligonucleotide construct of claim 77, wherein the modified nucleotide(s) of the functional sequence are independently selected to comprise the structure B-L-Func wherein B is independently selected from a pyrimidine or purine base; L is independently selected from a bond or a linker; and Func is a functional substituent.

79. The oligonucleotide construct of claim 77, wherein the functional element and coding element are part of a single linear or branched oligonucleotide.

80. The oligonucleotide construct of claim 77, wherein each of the functional element and coding element are attached to a support matrix.

81. The oligonucleotide construct of claim 77, wherein the functional element comprises the functional sequence flanked by two or more oligonucleotide sequences having partial or complete complementarity to each other.

82. The oligonucleotide construct of claim 77, wherein the coding element comprises the coding sequence flanked by two or more oligonucleotide sequences.

83. The oligonucleotide construct of claim 77, wherein the functional element and coding element are single stranded.

84. A library of oligonucleotide constructs according to claim 77, wherein the library comprises a plurality of oligonucleotide constructs having different sequence structure of the functional sequence.

85. A library of oligonucleotide constructs according to claim 84, wherein the oligonucleotide constructs differ in the base sequence of the functional sequence.

86. A method of preparing an oligonucleotide construct, the oligonucleotide construct comprising a functional element and a coding element, the method comprising: (i) extension of a functional element by addition of a single modified nucleotide to form a functional sequence, (ii) extension of a coding element by addition of a nucleotide n-mer to form a coding sequence, the n-mer selected to encode the structure of said modified nucleotide of step (i), wherein n is an integer of 2 or greater and the n-mer does not contain the modification of said modified nucleotide of step (i), wherein either step (i) or (ii) may be performed first and steps (i) and (ii) are performed sequentially and wherein steps (i) and (ii) are repeated as necessary to produce a coding sequence having a length n times the length of the functional sequence.

87. The method of claim 86, wherein extension of the coding element comprises addition of n nucleotide monomers.

88. The method of claim 86, wherein the oligonucleotide construct is a branched oligonucleotide, wherein the extension in (i) comprises addition of a single modified nucleotide to a branch moiety to form a first branch, and the extension in (ii) comprises addition of a nucleotide n-mer to the branch moiety to form a second branch.

89. The method of claim 86, wherein each oligonucleotide construct is attached to a solid support and preparation of the oligonucleotide constructs is conducted in accordance with a coding table that describes the relationship between each nucleotide added to the functional sequence and the n-mer added to the coding sequence at a corresponding position, wherein each performance of steps (i) and (ii) forms a round of extension, and wherein between rounds of extension the solid supports are mixed and then divided into 2x parts and at least one or each of the 2x parts is subjected to a further round of extension, where x is the number of n-mers in the coding table.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0393] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0394] FIG. 1. Diagram of an oligonucleotide construct attached to a support matrix during synthesis of a library on support matrix (herein referred to as a “Library on Support”). Functional element contains the functional sequence (black) and fixed sequences (white) and is attached to the support matrix via a spacer sequence. The coding element contains the coding sequence (diagonal line shading), coding-table ID sequence, fixed sequences and is attached to the support matrix via a spacer sequence.

[0395] FIGS. 2A, 2B and 2C. (A) Diagram of an oligonucleotide construct (Library on Support) attached to a support matrix during synthesis of the oligonucleotide construct. (B) Diagram of an oligonucleotide construct (Library on Support) attached to a support matrix during selection of effective oligonucleotide construct. (C) Diagram of a selected functional element.

[0396] FIG. 3. Diagram of an oligonucleotide construct attached to a support matrix during synthesis of a Library in Solution. Functional element contains functional sequence (black), fixed sequences (white) and is attached to a branch moiety via a spacer sequence. The coding element contains the coding sequence (diagonal line shading), coding-table ID sequence, and fixed sequences and is attached to the branch moiety via a spacer sequence. The oligonucleotide construct is attached to the support matrix via a cleavable linker attached to the branch moiety.

[0397] FIGS. 4A, 4B and 4C. (A) Diagram of an oligonucleotide construct (Library in Solution) attached to a support matrix during synthesis of the oligonucleotide construct. (B) Diagram of an oligonucleotide construct (Library in Solution) released from the support matrix during selection of effective oligonucleotide construct. (C) Diagram of a selected functional element.

[0398] FIG. 5. Diagram showing orientation of functional element, coding element and linker sequence for oligonucleotide construct according to Library in Solution. The functional and coding elements are each synthesised in 3′ to 5′ direction away from the branch moiety. Linker sequence is in 3′ to 5′ direction towards the branch moiety.

[0399] FIGS. 6A and 6B. (A) Illustration of an oligonucleotide construct having separate functional and coding elements attached to a support matrix. (B) Illustration of an oligonucleotide construct comprising a branched oligonucleotide attached to a support matrix, the branched oligonucleotide comprising a functional and coding elements.

[0400] FIGS. 7A, 7B, 7C and 7D. (A) 4,4′-dimethoxytrityl (DMTr) protected phosphoramidite. (B) Levulinoyl (Lev) protected phosphoramidite. (C) Illustration of a bead having a plurality of functional elements attached each protected with a 4,4′-dimethoxytrityl group and also having a plurality of coding elements attached each protected with a levulinoyl acid group. (D) Illustration of a bead having a plurality of branched oligonucleotide construct having a functional element protected with a 4,4′-dimethoxytrityl group and coding element protected with a levulinoyl group.

[0401] FIG. 8. Illustration of stepwise extension of functional and coding sequences and preparation of a combinatorial library. Extension of functional sequence with a modified nucleotide monomer is followed by extension of the coding sequence by addition of a non-modified nucleotide n-mer to form an oligonucleotide construct. Two protection groups (4,4′-dimethoxytrityl (DMTr) and Levulinoyl (Lev)) groups are used for synthesis of the oligonucleotide construct on a support matrix, one is used to protect the growing functional element and the other to protect the growing coding element. Serial de-protection and re-protection allows stepwise extension of the functional and coding elements. Mixing and re-splitting of support matrix of different constructs being simultaneously but separately prepared by the same process after one extension of each of the coding and functional sequences provides for formation of a combinatorial library of constructs having different functional sequences. The process illustrated can be repeated as many times as desired.

[0402] FIGS. 9A and 9B. (A) Illustration of one round of separate extension reactions of a plurality of different oligonucleotide constructs (top row), mixing of constructs (middle row) and splitting of constructs for a further round of extension (bottom row). (B) Coding table for n=2 illustrating the modified nucleotides (F.sub.x) that can be added to the functional sequence and corresponding unmodified nucleotide dinucleotide of the coding sequence.

[0403] FIGS. 10A, 10B and 10C. (A) Schematic illustration showing synthesis of linear/branched oligonucleotide construct on a support matrix followed by cleavage to generate a library of oligonucleotide constructs in solution. (B) Schematic illustration showing synthesis of linear/branched oligonucleotide construct on a support matrix in which the functional element and coding element are attached to a support matrix via branch moiety and cleavable/non-cleavable linker to generate a library on support matrices each attached to a different oligonucleotide construct. (C) Schematic illustration showing synthesis of oligonucleotide construct in which the functional element and coding element are each attached to a support matrix to generate a library of support matrices each attached to a different oligonucleotide construct.

[0404] FIGS. 11A and 11B. Coding tables showing relationship between coding sequence and functional sequence, where n=2. Modified nucleotides are indicated by reference to the base (A, C, G, T) and amino acid substituent by which it is modified (indicated in subscript using the amino acid three letter code). (A) Example of coding table for oligonucleotide constructs according to the present invention, e.g. dinucleotide CA in the coding sequence corresponds to C.sub.Phe (Cytosine modified by inclusion of an amino acid residue having the side chain of Phenylalanine) in the functional sequence. (B) Reference example of coding table adapted from U.S. Pat. No. 7,517,646.

[0405] FIGS. 12A and 12B. Coding tables showing relationship between coding sequence and functional sequence, where n=3. Modified nucleotides are indicated by reference to the base (A, C, G, T) and functional substituent by which it is modified (indicated in subscript using abbreviation appeared in this invention). (A) Example of coding table for oligonucleotide constructs according to the present invention, e.g. trinucleotide AAC in the coding sequence corresponds to C.sub.Phe (Cytosine modified by inclusion of an amino acid residue having the side chain of Phenylalanine) in the functional sequence. (B) Reference example of coding table adapted from U.S. Pat. No. 7,517,646.

[0406] FIG. 13. Synthesis scheme for preparation of deoxyuridine and deoxycytosine modified at the 5-position with an amino acid moiety substituent.

[0407] FIG. 14. Coding table of eight 5-position modified deoxyuridines and eight 5-position modified deoxycytosines, where n=2.

[0408] FIG. 15. Diagram illustrating alternative configurations of the functional element. A functional sequence flanked by fixed sequences allows formation of a hairpin loop structure. Insertion of additional fixed sequences within the functional sequence permits formation of more complex tertiary structures.

EXAMPLES

Example 1

[0409] Eight kinds of deoxycytidine (C.sub.1-8) and eight kinds of deoxyuridine (U.sub.1-8) are prepared each having a functional substituent at the 5-position of cytosine or uracil respectively (FIG. 13), in which the functional substituent corresponds to the side chain of an amino acid as follows: 1=Ala, 2=Asp, 3=Glu, 4=Gly, 5=Ile, 6=Lys, 7=Phe, 8=Ser.

[0410] Each of the 16 modified nucleosides are one-to-one related to a dinucleotide to form a coding table (FIG. 14).

Example 2—Preparation of Library in Solution

[0411] 1. A polymer solid support resin having free amino residue on its surface is conjugated with thymidine derivative Formula (B) by dehydration synthesis using HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) as described in Japan patent 5,365,005.

##STR00014## [0412] 2. The polymer solid support resin is packed in a universal column support. [0413] 3. Using a conventional DNA synthesizer and the phosphoramidite procedure, a DMT-thymidine-phosphoramidite is conjugated to the hydroxyl group of the solid support. [0414] 4. Glycerol derivative Formula (C) is conjugated with the hydroxyl group of thymidine on the solid support by conventional phosphoramidite method, (1) detritylation, (2) phosphoramidite activation, (3) coupling and capping, and (4) oxidation.

##STR00015## [0415] 5. DMT-deoxyadenine phosphoramidite, DMT-deoxyguanine phosphoramidite, DMT-deoxycytidine phosphoramidite and DMT-thymine phosphoramidite are used to synthesize a fixed sequence of the functional element from 3′ end to 5′ direction on DMT-protected hydroxyl group of the glycerol derivative. [0416] 6. Lev-deoxyadenine phosphoramidite, Lev-deoxyguanine phosphoramidite, Lev-deoxycytidine phosphoramidite and Lev-thymine phosphoramidite are used to synthesize a fixed sequence of the coding element from 3′ to 5′ direction on Lev-protected hydroxyl group of the glycerol derivative. [0417] 7. The resulting solid supports are removed from the universal support column and packed into 16 new universal support columns. [0418] 8. Each of the 16 columns are assigned for addition of 3 nucleotides relating to one cell of the coding table in FIG. 14, e.g. “A, A, U.sub.Ala”, “C, A, C.sub.Ala”, “A, C, U.sub.Glu”, “G, T, U.sub.Ser” etc., (underline=nucleotides added to coding sequence; bold=modified nucleotide added to functional sequence). For example, to add C.sub.Ala to the functional sequence and the encoding CA (5′ to 3′*) dinucleotide to the coding sequence (according to FIG. 14): C.sub.Ala phosphoramidite is added to the functional sequence by deblocking Lev with 0.5M diamine in pyridine/AcOH(1:1); then A is conjugated to the coding sequence by deblocking DMT with 3% TCA in Dichloromethane, and C is then conjugated to the coding sequence by deblocking DMT with 3% TCA in Dichloromethane. [*Note that synthesis by the phosphoramidite method is performed from the 3′-end to the 5′-end.] [0419] 9. After treating all 16 columns in the same way using an appropriate combination of nucleotides from each cell of the coding table, the solid supports from the universal support columns, mixed thoroughly, and packed into 16 new universal support columns. In this way, a library of oligonucleotide constructs each having a randomised functional sequence and corresponding coding sequence are prepared in accordance with the coding table. [0420] 10. Steps 7 and 8 are repeated as many times as necessary to obtain the desired length of functional sequence. [0421] 11. Where fixed sequence(s) are to be incorporated, DMT-deoxyadenine phosphoramidites (e.g. DMT-deoxyguanine phosphoramidite, DMT-deoxycytidine phosphoramidite and DMT-thymine phosphoramidite) are used to synthesise the fixed sequences of the functional element and Lev-phophoramidites are used for synthesis of the fixed sequence of coding element. [0422] 12. All protection groups including or excluding DMT and Lev groups are removed using deprotection reagent, such as a hindered base or aqueous ammonia, and cleaved from the solid support using an appropriate cleavage reagent, e.g. TCEP or DTT for a disulfide linker. [0423] 13. Purification and desalting are performed according to standard purification methods.

Example 3—Preparation of Library on Support

[0424] 1. A polymer solid support resin having a free hydroxide residue on its surface is packed in a universal column support. [0425] 2. Using a conventional DNA synthesizer and the phosphoramidite procedure, a DMT-thymidine-phosphoramidite is conjugated to a hydroxyl group of the solid support four times so that a thymidine tetranucleotide is tethered on the solid support. [0426] 3. After removing DMT by 3% TCA in Dichloromethane, a mixture of DMT-thymine phosphoramidite and Lev-thymine phosphoramidite are conjugated to said thymidine tetraoligonucleotide. [0427] 4. By following the steps 4 to 10 of Example 2 (“Library in Solution” preparation), fixed sequences, functional sequences and coding sequence are synthesized. [0428] 5. All of the protection groups including DMT and Lev groups are removed using deprotection reagents as indicated in Example 2. [0429] 6. The resulting “Library on Support” is recovered from the universal support column.

Example 4—Identification of Oligonucleotide Constructs Binding to a Target Molecule from a Library in Solution

[0430] Using the Library in Solution (“Library”) from Example 2, oligonucleotide constructs that bind with high affinity to a protein target are identified.

[0431] A His-tagged PSA (prostate specific antigen) protein (the target molecule) is immobilized on His-Mag Sepharose-Ni bead to immobilize the protein. A suspension of the beads is mixed with the Library solution.

[0432] Unbound components in the Library are removed by washing sufficiently with a buffer solution containing high concentration of salt and/or low concentration of mild detergent. After washing, target molecule:oligonucleotide complex is eluted from the His Mag Sepharose-Ni resin by 500 mM imidazole solution.

[0433] The recovered target molecule:oligonucleotide complex is subjected to PCR amplification of coding sequence using two primer oligonucleotides complementary to the fixed sequences located at 5′ and 3′ ends of the coding sequence, respectively. The PCR amplified DNA fraction is subjected to conventional DNA sequencing to obtain sequence information for the coding element, including the coding sequence, and coding table ID sequence.

[0434] The coding sequence is translated using the coding table to determine the sequence structure (the base sequence and associated amino acid moiety modifications) of the functional sequence.

[0435] Functional sequence oligonucleotide molecules, with or without additional nucleotides providing other functions such as site-specific attachment site, labeling with fluorescent dye and biotinylation, are synthesised by the phosphoramidite method according to the determined sequence structure of the functional sequence. The newly synthesised oligonucleotides are re-assayed for binding to PSA using Surface Plasmon Resonance measurement, sandwich assay and/or affinity column profiling. Varying the conditions for target binding allows selection of functional sequences that exhibit the highest affinity for PSA.

[0436] After PCR amplification of target molecule:oligonucleotide complex, the sample is subjected to mild basic conditions to detach the complex, the oligonucleotide fraction is recovered by ethanol precipitation and reapplied for affinity selection described above. This provides a second batch of PCR product for sequencing.

[0437] By comparing occurrence of sequences between the first batch, second batch and further re-selection of PCR products, the enrichment process is verified and allows identification of potent candidate sequences.

Example 5—Identification of Oligonucleotide Constructs Binding to a Target Molecule from a Library on Support

[0438] Using the “Library on Support” (“Library”) from Example 3, oligonucleotide constructs that bind with high affinity to a protein target are identified.

[0439] PSA (prostate specific antigen) protein (the target molecule) is conjugated with fluorescent dye such as 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole before starting selection process.

[0440] Labeled PSA is mixed with suspension of Library overnight. After recovering and washing solid support fraction by filtration, solid support fraction is diluted to optimum concentration for cell sorting equipment (or bead sorting equipment) and each single solid support having stronger fluorescent intensity than threshold is fractionated for recovery.

[0441] Each single solid support recovered by sorting equipment is applied for PCR amplification of the coding sequence using two primer oligonucleotides complementary to the fixed sequences located at 5′ and 3′ ends of the coding sequence, respectively. PCR amplified DNA fraction is subjected to conventional DNA sequencing to obtain sequence information for the coding element, coding sequence, and coding table ID sequence.

[0442] The resulting coding sequence is translated using the coding table to determine the sequence structure (the base sequence and associated amino acid moiety modifications) of the functional sequence.

[0443] Functional sequence oligonucleotide molecules, with or without additional nucleotides providing other functions such as site-specific attachment site, labeling with fluorescent dye and biotinylation, are synthesised individually by the phosphoramidite method according to the determined sequence structure of the functional sequence. The newly synthesised oligonucleotides are re-assayed for binding to PSA using Surface Plasmon Resonance measurement, sandwich assay and/or affinity column profiling. Varying the conditions for target binding allows selection of functional sequences that exhibit the highest affinity for PSA.

[0444] Since one particle of the solid support, e.g. one bead, may have hundreds of thousands of functional elements attached to it, a corresponding number of target molecules may be bound to a single particle. The intensity of fluorescence therefore reflects the statistical value of the result and may be used to characterise the functional element in terms of ability of the functional sequence to contribute to target molecule binding.