(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`
`International Bureau
`
`(43) International Publication Date
`19 September 2002 (19.09.2002)
`
`pCT
`
`(10) International Publication Number
`w() 02/072791 A2
`
`(51) International Patent Classification7:
`
`C12N (74) Agents: CARROLL, Peter, G. et a1.; Medlen & Carroll,
`LLP, Suite 350, 101 Howard Street, San Francisco, CA
`
`(21) International Application Number:
`
`PCT/Uso2/08171
`
`94105 (US)-
`
`(22) International Filing Date:
`
`14 March 2002 (14.03.2002)
`
`(25) Filing Langm‘ge’
`
`(26) Publication Language:
`
`EngliSh
`
`English
`
`(30) Priority Data:
`60/275,666
`10/099,382
`
`14 March 2001 (14.03.2001)
`13 March 2002 (13.03.2002)
`
`US
`US
`
`(71) Applicant 0’0r all designated States except US): THE
`REGENTS OF THE UNIVERSITY OF MICHIGAN
`[US/US J; 3003 South State Street, Ann Arbor, Ml 48109
`(US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`
`VN, YU, ZA’ ZM, ZW'
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`
`Eurasian patent (AM’AZ’BYv KG,KZ,MD,RU,TJ,TM),
`European Pate“I (AT, BE, CH, CY DE, DK, ES FT, FR,
`GB GR IE IT LU MC NLs PT SE: TR) OAPI Pawnt
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Gw, ML, MR,
`NE, SN, TD, TG).
`
`(72) Inventors; and
`Published:
`(75) Inventors/Applicants (for US only): GOA, Xiaolian
`[US/US]; 221113 Bellafontaine Blvd., Houston, TX 77030 i without international search report and to be republished
`(US). ZHOU, Xiaochuan [CN/US]; 2212 B Bellefontaine
`"P0" receipt Off/1at report
`Blvd., Houston, TX 77030 (US). YU, Peilin [CN/US];
`6245 Renwiek Drive, #4210, Houston, TX 77081 (US).
`ZHANG, Hua [CN/US]; 6924 Stella Link Road, #19,
`Houston, TX 77025 (US). LEPROUST, Erie [FR/US];
`275 Union Avenue, #F—ll24, Campbell, CA 95008 (US).
`XIANG, Qin [CN/US]; Houston, TX 77030 (US). PEL-
`LOIS, Jean Phillipe [FR/US]; 5500 N. Braeswood, #234,
`Houston, TX 77096 (US).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andAbbreviations”appearing at the begin—
`ning ofeach regular issue ofthe PCT Gazette.
`
`1A2 (54) Title: LINKERS AND COeCOUPLING AGENTS FOR OPTIMIZATION OF OLIGONUCLEOTIDE SYNTHESIS AND PUe
`
`ON RIFICATION ON SOLID SUPPORT
`
`WO02/0727
`
`(57) Abstract: A method of modulation of synthesis capacity on and cleavage properties of synthetic oligomers from solid support
`is described. The method utilizes linker molecules attached to a solid surface and co—coupling agents that have similar reactivities
`to the coupling compounds with the surface functional groups. The preferred linker molecules provide an increased density of poly—
`mers and more resistance to cleavage from the support surface. The method is particularly useful for synthesis of oligonucleotides,
`oligonucleotides microarrays, peptides, and peptide microarrays. The stable linkers are also coupled to anchor molecules for synthe—
`sis of DNA oligonucleotides using on support purification, eliminating time—consuming chromatography and metal cation presence.
`oligonucleotides thus obtained can be directly used for mass analysis, DNA amplification and ligation, hybridization, and many
`other applications.
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`Linkers and CoeCoupling Agents for Optimization of Oligonucleotide Synthesis and
`
`Purification on Solid Supports
`
`Field of the Invention
`
`The instant disclosure pertains to a method for optimization of synthesis and purification
`
`of synthetic oligomers, such as oligonucleotides and peptides, on a solid support. In particular,
`
`the disclosure pertains to the use of linkers and co—coupling agents for synthesizing
`
`oligonucleotides in a controlled manner and for obtaining oligonucleotides of high quality using
`
`simple purification procedures. The method particularly relates to high throughput synthesis of
`
`oligonucleotides for a variety of applications.
`
`Background of the Invention
`
`The growing importance of combinatorial synthesis has created a need for new resins and
`
`linkers having chemical and physical properties to‘accornmodate a Wide range of conditions,
`
`since success depends on the ability'to synthesize diverse sets of molecules on solid supports and
`to then cleave those molecules from the supports cleanly and in good yield.
`
`Parallel synthesis, miniaturized analysis and interrogation of libraries of molecules are
`
`being perceived'as one the most promising approaches available to modern chemistry and w
`
`biology (Gallop et al., (1994) J Med. Chem. 37, 1233-1251; Gordon et al.,(l994) J. Med. Chem.
`
`37, 1385—1401; Ellman et a1., (1997) Proc. Natl. Acad. Sci. USA, 94, 2779—2282; Lebl, M. (1999)
`
`J. Comb. Chem. 1, 3‘24. Examples include applications in combinatorial synthesis and screening ‘
`
`of pharmaceutical compounds, biomolecular assays, and gene analysis using Oligonucleotide
`
`microarrays or DNA chips. A common platform for these micro—chemical and biological
`
`experiments is planar surfaces, such as those made from silicon-based materials or synthetic
`
`polymers. Among these, glass plates (e.g. microscope slides, Which are borosilicate glass) are
`
`easily available, easy to handle, and commonly used.
`
`Linkers are molecules that can be attached to a solid support and to which the desired
`
`members of a library of chemical compounds may in turn be attached. When the construction of
`
`the library is complete, the linker allows clean separation of the target compounds from the solid
`
`support without harm to the compounds and preferably without damage to the support. Several
`
`linkers have been described in the literature. Their value is constrained by the need to have
`
`sufficient stability to allow the steps of combinatorial synthesis under conditions that will not
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`cleave the linker, while still being cleavable under at least one set of conditions that is not
`
`employed in the synthesis. For example, if an acid labile linker is employed, then the
`
`combinatorial synthesis must be restricted to reactions that do not require the presence of an acid
`
`of sufficient strength to endanger the integrity of the linker. This sort of balancing act often
`
`imposes serious constraints on the reactions that can be employed in preparing the library.
`
`Accordingly, what needed in the art are improved reagents for facilitating the synthesis
`
`and purification of polymers on solid supports.
`
`Summary of the Invention
`
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`
`In some embodiments, the present invention provides a stable linker [and more
`
`particularly, a selectively cleavable linker, i.e. a linker that is cleavable under at least one set of
`
`chemical reaction conditions, while not being substantially cleaved (i.e. approximately 90% or
`
`greater remains uncleaved) under another set (or other sets) of reaction conditions] for polymer
`
`synthesis comprising a chemical moiety immobilized on a solid support and not substantially
`
`cleaved under polymer synthesis conditions, which may include chain growth and even removal
`of the protecting groups from the polymer chain. A linker group typically has two ends, wherein
`
`one of the ends comprises a substrate attaching group and wherein the other of the ends
`
`comprises a polymer attaching group, wherein the polymer attaching group is preferably
`
`20
`
`covalently linked to an anchor moiety and the anchor group has an attaching group for polymer
`synthesis. The present invention is not limited to any particular linker group. Indeed, the use of
`
`a variety of linker groups is contemplated, including, but not limited to, alkyl, ether, polyether,
`
`alkyl amide groups or a combination of these groups. The present invention is not limited to the
`
`use of any particular alkyl group. Indeed, the use of a variety of alkyl groups is contemplated,
`
`including —(CH2)n—, wherein n is from about 4 to about 20. The use of a variety of ether and
`
`polyether groups is contemplated, including ~(OCH2CH2)n—, wherein n is from about 1 to about
`
`20. The use of a variety of alkyl amide groups is contemplated, including —(CH2)m—C(O)NH—
`
`(CH2)n—and —(OCH2CH2)mvC(O)NH—(OCH2CH2)n—, wherein m and n can be the same or
`
`different and m and n are from about 1 to about 20. The use of a variety of amide groups having
`
`the linking units of alkyl or ether bonds is contemplated, including —-R1—C(O)NH—R2—, wherein
`
`R1 and R2 are alkyl, ether, and polyether groups.
`
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`
`The present invention is not limited to the use of any particular substrate attaching group.
`
`Indeed, the use of a variety of substrate attaching groups is contemplated, including, but not
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`limited to trichlorosily] and trialkyloxysilyl functional groups. The present invention is not
`
`limited to the use of any particular polymer attaching group. Indeed, the use of a variety of
`
`polymer attaching groups is contemplated, including, but not limited to amine, hydroxyl, thiol,
`
`carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate groups.
`
`In preferred embodiments of the present invention, the linker is covalently bound to a
`
`support. The present invention is not limited to any particular support. Indeed, the use of a
`
`variety of supports is contemplated, including, but not limited to polymerized Langmuir Blodgett
`
`film, functionalized glass, Si, Ge, GaAs, GaP, SiOz, SiN4, modified silicon, polyacrylamide,
`
`polytetraflouroethylene, polyvinylidendiflouride, polystyrene, polycarbonate, and co—polymers.
`
`The present invention is not limited to the use of any particular anchor moiety. Indeed,
`
`the use of a variety of anchor moieties is contemplated, including, but not limited to, those of the
`
`following 1,2—diol derivatives of structures shown below:
`
`qu0
`
`P1WO
`
`|
`
`:0:1 B]
`p2
`
`R 000 or
`1()
`
`:o% B,
`P‘IO
`OC(O)R1
`2
`
`P1WO
`
`P1WO
`
`| F0; B;
`
`OR 0.
`
`p2
`
`E0:13;
`PIo
`OR
`2
`
`Wherein P1 and P2 are chain units comprised of polymer or linker and polymer; B is a
`
`nucleobase; R1 are substitution groups, such as CH3, R2P11(R2 are substitution groups on the
`
`phenyl ring, such as SCH3, Cl, N02), CHZCHZCN. R is a protecting group, Which is OC(O)R1, t-
`
`butyldimethylsilyl (TBDMS), or other protecting groups used for 2’- or 3’-O protection of
`
`ribonucleotides. Once the protecting group is removed, the adj acent OH can accelerate the
`
`hydrolysis of the phosphodiester bond, resulting in cleavage of the polymer chain.
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`The present invention is not limited to the use of any particular anchor moiety. Indeed,
`
`the use of a variety of anchor moieties is contemplated, including, but not limited to, those of the
`
`2’—deoxyuridine (dU) and abasic moiety of structures shown below:
`
`0
`
`Pi
`
`P1w0
`
`| :0:
`o
`Pi
`
`,40H
`
`abasic moiety
`ribose form
`
`P1""0
`5
`
`Y
`0
`P:
`
`0H {0
`[3
`
`abasic moiety
`aldehyde form
`
`Wherein P1 and P2 are chain units comprised of linker and polymer or polymer; dU in an
`
`10
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`oligonucleotide that can be incorporated as its phosphoramidite and selectively cleaved by uracil-
`
`DNA glycosylase (UDG) (from E. coli), Which catalyzes the removal of uracil from single— and
`
`double—stranded DNA. The apyriminic or the abasic sites formed by UNG are susceptible to
`
`cleavage by heat under alkaline conditions. The abasic moiety can be incorporated as its
`
`phosphoramidite monomer and is labile under basic conditions; treatment using amines, such as
`piperidine, EDA, and N,N’—dimethy1ethylenediamine causes [3— or B~ and 5—eliminations to give
`
`15
`
`5 ’—phosphate and 3 ’—phosphate or other 3’—products.
`
`The present invention also includes anchor moieties of ribose nucleotides that can be
`
`incorporated in regular DNA synthesis using their phosphoramidites. These residues can be
`
`cleaved by ribonucleases, such as RNases A (cutting mostly pyrimidines), T1 (cutting mostly
`
`20
`
`G’s) and U2 (cutting mostly A’s). The 3’— and 5’—ends of the cleaved sequences may require
`
`further modification using chemical and enzymatic conditions to obtain sequences with 3’— and
`5’-fucntiona1 groups required by the subsequent applications. There are many reactions
`
`conditions available for these modifications, including using 5 ’~ or 3’-exonucleases for removal
`
`of terminal phosphate group.
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`The present invention is not limited to the use of any particular anchor moiety. Indeed,
`
`the use of a variety of anchor moieties is contemplated, including, but not limited to, those of the
`
`2’—deoxyuridine (dU) and abasic moiety of structures shown below:
`
`The present invention is not limited to the use of any particular anchor moiety. Indeed,
`
`the use of a variety of anchor moieties is contemplated, including, but not limited to, those of the
`
`modified nucleotides of structures shown below:
`
`P3WO
`
`I
`
`0 B
`
`R O..P4
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`20
`
`Wherein P3 and P4 are chain units comprised ofpolymer or linker and polymer; one or
`
`both P3 and P4 chains are linked to the nucleotide through thioate phosphate (PS) bonds. The PS
`
`bond forms in regular DNA or RNA chemical synthesis when the oxidation step employs either
`
`tetraethylthiuram disulfide (TETD) or 3H—1, 2—bensodithiol—3—one 1, 1—dioxide (BDTD) for
`
`sulfurizing phosphite trimesters formed from coupling of phosphoramidites (Spitzer, S.;
`
`Eckstein, F. (198 8) Nucleic Acids. Res. 16, 11691—11704). The PS linkage can be selectively
`
`cleaved by the addition of 12 (Strobel, S. A., and Shetty, K. Proc. Natl. Acad. Sci. USA. 94,
`2903-2908).
`I
`
`In preferred embodiments, the anchor moiety is stable under conditions used for polymer
`
`synthesis, which may include conditions for chain grth as well as conditions for removal of
`
`the protecting groups from the polymer chain. The anchor moieties of the present invention may
`
`be cleavable under certain selected conditions. The present invention is not limited to any
`
`particular set of selective cleavage conditions. Indeed, the present invention contemplates that a
`
`variety of cleavage conditions may be utilized when appropriate, including 2—OH assisted 1—
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`phosphate hydrolysis and enzymatic cleavage of the chemical bonds. In other embodiments of
`the present invention, the anchor moiety includes a polymer attaching group. In still further
`
`embodiments, a polymer is attached to the anchor moiety. The present invention is not limited to
`
`any particular polymer. Indeed, a variety of polymers are contemplated, including, but not
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`limited to peptides and oligonucleotides.
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`The present invention is not limited to the use of any particular anchor moiety. Indeed,
`
`the use of a variety of anchor moieties is contemplated, including, but not limited to, those of the
`
`following structure:
`
`it
`L—o—P—o
`|
`O—
`
`o
`
`O
`
`OCOR o
`
`O(CH3)CN
`I
`\fi_____0_ P'
`
`O
`
`wherein L is the linker and P’ is a polymer.
`
`In still other embodiments, the present invention provides compounds possessing the
`
`structure:
`
`Rs—L— Rp
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`20
`
`wherein RS is a substrate attaching group, Rp is a polymer attaching group, and L is the linker.
`
`The present invention is not limited to the use of any particular substrate attaching group
`
`(R5). Indeed, the use of a variety of substrate attaching groups is contemplated, including, but
`
`not limited to chlorosilyl and alkyloxysilyl functional groups. The present invention is not
`
`limited to the use of any particular polymer attaching group. Indeed, the use of a variety of
`
`polymer attaching groups is contemplated, including, but not limited to amine, hydroxyl, thiol,
`
`carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate groups.
`
`In some embodiments, Rp is selected from the group including, but not limited to amine, ,
`
`hydroxyl, thiol, carboxylic acid, ester, amide, epoxide, isocyanate, and isothiocyanate groups.
`
`In some embodiments of the present invention, the linker is covalently bound to a
`
`support. The present invention is not limited to any particular support. Indeed, the use of a
`
`variety of supports is contemplated, including, but not limited to polymerized Langmuir Blodgett
`
`film, functionalized glass, Si, Ge, GaAs, GaP, SiOz, SiN4, modified silicon, polyacrylamide,
`
`polytetraflouroethylene, polyvinylidendiflouride, polystyrene, and polycarbonate.
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`
`In still further embodiments, the present invention provides methods for synthesizing
`oligonucleotides comprising: providing a substrate; a plurality of stable linkers; a plurality of
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`anchor moieties; and nucleotide monomers; derivitizing the substrate with the plurality of stable
`
`linkers; attaching the anchor moieties to the stable linkers; and synthesizing oligonucleotides on
`
`the plurality of anchor moieties. In some embodiments, the methods further comprise
`
`deprotecting the oligonucleotides and selectively cleaving the oligonucleotides from the substrate
`
`by reacting the substrate under conditions such that the polymer is cleaved at the anchor moiety.
`
`In still further embodiments, the present invention provides methods for controlling the
`
`number of oligonucleotides synthesized at a predetermined site on a substrate comprising:
`providing a substrate; a plurality of stable linkers; a plurality of anchor moieties; nucleotide
`
`monomers; and co—coupling agents; derivitizing the substrate with said plurality of stable linkers;
`
`attaching the anchor moieties to the stable linkers; and synthesizing a oligonucleotide on the
`
`plurality of anchor moieties from the monomers in the presence of the co—coupling agents under
`
`conditions such that at least a portion of the oligonucleotides are terminated.
`
`In still other embodiments, the present invention provides methods of purifying
`
`oligonucleotides comprising: providing: a substrate comprising oligonucleotides attached to a
`
`substrate via an anchor moiety attached to a stable linker group, a deprotecting solution, and a
`
`wash solution; deprotecting said oligonucleotides with said deprotecting solution, washing said
`
`oligonucleotides attached to a substrate with said wash solution, and cleaving said
`
`oligonucleotides at said anchor group to provide purified oligonucleotides, wherein said purified
`
`oligonucleotides are characterized by the substantial absence of metal ions and/or other
`
`contaminants and said stable linker group remains attached to said substrate.
`
`In still other embodiments, the present invention provides methods of obtaining purified
`
`oligonucleotides comprising: providing: a substrate comprising oligonucleotides attached to a
`
`substrate via an anchor moiety attached to a stable linker group, a deprotecting solution, a wash
`
`solution, and a cleavage solution; deprotecting said oligonucleotides with said deprotecting
`
`solution, washing said oligonucleotides attached to a substrate with said wash solution, and
`
`cleaving said oligonucleotide using said cleavage solution at said anchor group to provide
`
`purified oligonucleotides, wherein said purified oligonucleotides are characterized by the
`substantial absence ofmetal ions and said stable linker group remains attached to said substrate.
`
`The oligonucleotides thus obtained have many applications, such as as substrates of nucleases,
`
`polymerases, kinases, or ligases, known to those of skilled in the art.
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`Description of the Figures
`
`‘
`
`Figure 1 provides examples of the chemical structure of the linker groups of the present
`
`invention attached to a solid substrate.
`
`Figure 2 provides exemplary chemical structures for a fluorescein tag, a chain terminator
`
`(co-coupling agent) and an anchor.
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`Figure 3 displays electrophoresis gel profiles of T10 cleaved from glass plates at 15, 30
`
`and 60 min upon treatment with cone. aq. NH40H. The T10 with amide linker is shown on the
`
`left panel and the T10 with C8 linker is shown on the right panel.
`
`Figure 4 presents results of an assay of oligonucleotide synthesis using a termination
`
`nucleophosphorainidite, 5’—MeO-T, to probe the presence of available sites for coupling with a
`
`phosphoraniidite at different reaction stages.
`
`(A) Regular T3 synthesis on glass plates. (B)
`
`Illustration of the use of termination monomer. T on glass plate is coupled with MeO—T, resulting
`
`in the formation of a terminated dimer T—T(OMe), which can not undergo further chain growth.
`
`(C) Illustration of the hypothesis for reaction with more hindered surface sites in several
`
`continued reaction cycles. (D) 32P—gel electrophoresis analysis of the experiments using the
`
`termination 5’—MeO—T at different stages of oligonucleotide synthesis.
`
`Figure 5 presents a comparison of the probe sequences synthesized using the amide and
`
`C8 linkers and used for three time hybridization experiments.
`
`Figure 6 presents a schematic depiction of the synthesis of an exemplary anchor moiety.
`
`Figure 7 presents a schematic depiction of the synthesis of an exemplary oligonucleotide.
`
`Figure 8 presents a schematic depiction of deprotection and cleavage of an exemplary
`
`oligonucleotide.
`
`Figure 9 presents HPLC data for oligonucleotides synthesized on the supports of the
`
`25
`
`present invention.
`
`Figure 10 presents HPLC data for the enzyme purified sequence cleaved from CPG.
`
`Figure 11 presents the results of PCR experiments conducted with oligonucleotide
`
`primers synthesized on the supports of the present invention.
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`Definitions
`
`The following terms are intended to have the following general meaning as they are used
`
`herein:
`
`The term “substrates” and “solid supports” are used interchangeably to refer to any
`
`material that is suitable for derivatization with a linker group. Examples of substrates include,
`
`but are not limited to glass, Si—based materials, functionalized polystyrene, flinctionalized
`
`polyethyleneglycol, functionalized organic polymers, nitrocellulose or nylon membranes, paper,
`
`cotton, and materials suitable for synthesis. Solid supports need not be flat. Supports include
`
`any type of shape including spherical shapes (e.g., beads). Materials attached to solid support
`
`may be attached to any portion of the solid support (e.g., may be attached to an interior portion
`
`of a porous solid support material). Preferred embodiments of the present invention have
`
`biological molecules such as oligonucleotides and peptides attached to solid supports. A
`
`compound is “attached” to a solid support when it is associated with the solid support through a
`
`non—random chemical or physical interaction. In some preferred embodiments, the attachment is
`
`through a covalent bond.
`
`As used herein, the terms “linker” and “linker group” are used interchangeably to refer to
`
`chemical moieties that are attachable to a solid support on one end and an anchor group or
`
`polymer on the other end. The "linker" and “linker group” are atoms or molecules that link or
`
`bond two entities (e.g., solid supports, oligonucleotides, or other molecules), but that is not a part
`
`of either of the individual linked entities. In general, linker molecules are oligomeric chain
`
`moieties containing 1—200 linearly connected chemical bonds. One end of a linker chain is
`
`immobilized on substrate surface, such as through —SiO- bond formation. The other end of a
`
`linker chain contains a functional group that can be converted to an OH or an NH; group.
`
`Examples of linkers include, but are not limited to the chemical moieties shown in Figure l and
`
`—(OCH2CH2)H:, wherein n is from about 1 to about 20. The use of a variety of alkyl amide
`
`groups is contemplated, including —(CH2)m~C(O)NH~(CH2)n—and —(OCH2CH2)m—C(O)NH—
`
`(OCHgCH2)n—, wherein m and n can be the same or different and m and n are from about 1 to
`
`about 20. The use of a variety of amide groups having the linking units of alkyl or ether bonds is
`
`contemplated, including —R1——C(O)NH—R2—~, wherein R1 and R2 are alkyl, ether, and polyether
`
`groups. Linkers can have substitutions to have branched chain structures, such as dendritic
`
`structures. Multiple linkers can be covalently connected to form an extended linker chain.
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`The term “anchor group or moiety” refers to a chemical moiety that connects a linker and
`
`a synthesized oligonucleotide or other polymer and which can be selectively cleaved to release
`
`oligonucleotides or other polymers from substrate surface. For example, the anchor may include
`
`the structure —C(X)~C(Y)— (X may be OPOZO~oligonucleotide), (Y is a functional group that may
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`function as a nucleophile, for example, Y may bean OH, NHz or SH). Preferably, the ~C(X)—
`C(Y)- is part of a ring moiety and further a five member ring moiety. The anchor may include
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`dU, abasic group, ribonucleotides, thioate phosphodiester, when incorporated into
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`oligonucleotides, which can selectively cleaved by treatment with specific enzymatic digestion
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`or chemical degradation conditions.
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`The term “protected nucleotides” refers to nucleotides containing nucleobase protecting
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`groups, such as 4—NH—benzol in cytidine and adenine and Z-NH—isobutyryl in guanosine, sugar
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`protecting groups, such as 2'—O—t—butyldimethylsilyl in ribonucleotides, and phosphate protecting
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`groups, such as P-O—(2-cyano)ethylphosphine, etc. “Protecting group” refers to a molecule or
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`chemical group that is covalently attached to a moiety of a compound to prevent chemical
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`modification of the moiety of the compound or modification of specific chemical groups of the
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`compound. For example, protecting groups may be attached to a reactive group of a compound
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`to prevent the reactive group from participating in chemical reactions including, for example,
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`intramolecular reactions. In some cases, a protecting group may act as a leaving group, such that
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`when the molecule is added to another compound in a desired synthesis reaction, the protecting
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`group is lost, allowing a reactive group to participate in covalent bonding to the compound. The
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`phosphoramidites of the present invention typically contain one or more protective groups prior
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`to their addition to nucleic acid molecules. For example, the reactive phosphate of the
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`phosphoramidite (i.'e., the phosphate group that is covalently attached to another molecule when
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`the phosphoramidite is added to the other molecule) may contain one or more protecting groups.
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`A detailed description of phosphoramidites and their addition to nucleic acid molecules is
`provided Beaucage and Iyer (Tetrahedron 4921925 [1993]), herein incorporated by reference in
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`its entirety.
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`As used herein, the term “stable”, when used in reference to a linker or an anchor group,
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`refers to a property of the compound or the chemical moiety which is not cleaved by certain
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`reactions conditions, but selectively cleavable by different reaction conditions. These orthogonal
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`reactions are well established in solid phase synthesis. The present invention is not limited to any
`
`particular set of selective cleavage conditions. Indeed, the present invention contemplates that
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`the siloxane linkers are stable under anhydrous ethylene diamine treatment, but a variety of
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`cleavage conditions may be utilized when appropriate, including base hydrolysis of the Si—O
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`bond. Further, the present invention contemplates that the the 1,2-diol anchors are stable to basic
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`hydrolysis when one of the OH group is protected with at protecting moiety, but a variety of
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`cleavage conditions may be utilized after the OH protecting group is removed, including 2—OH
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`assisted l-phosphate hydrolysis under basic conditions. Thus, the present invention
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`contemplates in one embodiment linkers and/or anchor groups that are stable to basic hydrolysis.
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`In another embodiment, the present invention contemplates linkers and/or anchor groups that are
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`stable to acid hydrolysis.
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`As used herein, the term “selective cleavable”, when used in reference to a linker or an
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`anchor group, refers to a property of the compound or the chemical moiety is not cleaved by
`
`certain reactions conditions, but selectively cleavable by different reaction conditions. These
`
`orthogonal reactions are well established in solid phase synthesis. The present invention is not
`
`limited to any particular set of selective cleavage conditions. Indeed, the present invention
`
`contemplates that the siloxane linkers are stable under anhydrous ethylene diamine treatment, but
`
`a variety of cleavage conditions may be utilized when appropriate, including base hydrolysis of
`
`the Si—O bond. Further, the present invention contemplates that the 1,2—diol anchors are stable to
`
`basic hydrolysis when one of the OH group is protected with a protecting moiety, but a variety of
`
`cleavage conditions may be utilized after the OH protecting group is removed, including 2—OH
`
`assisted l—phosphate hydrolysis under basic conditions. Further, the present invention
`
`contemplates that dU, abasic moiety, ribonucleotides, and thioate phosphodiester are stable under
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`regular DNA or RNA synthesis conditions but may be selectively cleaved by specific chemical
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`or enzymatic treatments.
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`As used herein, the term “substrate attaching group” refers to any chemical group that is
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`useful for attaching a linker to a substrate. Examples of substrate attaching groups include, but
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`are not limited to, monochlorosilyl, monoalkoxysilyl, trichlorosilyl or trialkoxysilyl groups.
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`As used herein, the term “polymer attaching group” refers to a functional group or groups
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`that can be converted to a functional group, for example, an OH or an NH; group, that is used for
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`initiating synthesis of a polymer on a linker or attaching an anchor moiety to a linker. Examples
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`of polymer attaching groups include, but are not limited to, amino, hydroxy, thiol, carboxylic
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`acid, ester, amide, isocyanate or isothiocyanate group, most preferably an OH or a NH2 group.
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`Methods for such functionalization are well known in the art (See, e.g., Bigley et al., J. Chem.
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`Soc. CB):1811—18 (1970).
`
`‘
`
`As used herein, the term “synthesis initiation site” refers to a chemical group on a linker
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`or an anchor moiety or any other chemical entity that is used as a site for initiating synthesis of a
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`polymer chain.
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`As used herein, the term “spacer” refers to a chemical group connected to a linker or an
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`anchor moiety that is used to extend the length of the linker moiety and as a site for initiating
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`synthesis of a polymer chain. Examples of spacer include, but are not limited to, ethyleneglycol
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`polymer, alkyl, oligonucleotides, peptides, peptditomimetics.
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`The term "oligonucleotide" as used herein is defined as a molecule comprising two or
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`more deoxyribonucleotides or ribonucleotides, preferably at least 4 nucleotides, more preferably
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`at least about 10—15 nucleotides and more preferably at least about 15 to 200 nucleotides. The
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`exact size will depend on many factors, which in turn depend on the ultimate function or use of
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`the oligonucleotide. The oligonucleotide may be generated in any mamier, including chemical
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`synthesis, DNA replication, reverse transcription, PCR, ligation, or a combination thereof.
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`Because mononucleotides are reacted to make oligonucleotides in a manner such that the
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`5‘ phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in
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`one direction Via a phosphodiester linkage, an end of an oligonucleotide is referred to as the "5'
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`end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the
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`"3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose
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`ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also
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`may be said to have 5' and 3' ends. A first region along a nucleic acid strand is said to be
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`upstream of another region if the 3' end of the first region is before the 5' end of the second
`
`region when moving along a strand of nucleic acid in a 5' to 3' direction. An oligonucleotide
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`sequence is written in 5’- to 3’ direction by convention.
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`As used herein, the term “co—coupling agent” refers to a compound which when
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`incorporated into a polymer serves as chain terminator, i.e. terminating the chain growth. The co-
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`coupling agent preferably has a structure similar to the monomers used in the polymer synthesis
`
`reaction. The co-coupling agent can be mixed with coupling agent in the synthesis, resulting in a
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`mixture of extendible and non-extendible sequences that n