WO2010151714A2 - Molecular arrays - Google Patents

Molecular arrays Download PDF

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Publication number
WO2010151714A2
WO2010151714A2 PCT/US2010/039882 US2010039882W WO2010151714A2 WO 2010151714 A2 WO2010151714 A2 WO 2010151714A2 US 2010039882 W US2010039882 W US 2010039882W WO 2010151714 A2 WO2010151714 A2 WO 2010151714A2
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Prior art keywords
anchor
spacer
nucleic acid
moiety
array
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PCT/US2010/039882
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French (fr)
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WO2010151714A3 (en
Inventor
Zhaohui Zhou
George A. Fry
Sam Lee Woo
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Life Technologies Corporation
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Publication of WO2010151714A3 publication Critical patent/WO2010151714A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the disclosed embodiments are generally directed to compositions, systems and methods for producing arrays of single molecules immobilized at discrete locations on a solid surface.
  • the spacing between adjacent immobilized molecules is tunable and can be adjusted.
  • the disclosure relates to arrays of nucleic acid molecules which are immobilized at locations on a solid surface, which can optionally have tunable spacing distances between the nucleic acid molecules, as well as to methods, compositions and systems for preparing such arrays.
  • Single molecule arrays are used routinely for a wide range of laboratory procedures for diagnostic, screening, and characterization analyses, including polymorphism analyses, elucidation of protein/nucleic acid interactions, screening of candidate pharmaceutical compounds, and sequencing.
  • Preparing such arrays typically involves depositing single molecules onto a solid or semi-solid surface in a random, semi-ordered or ordered pattern.
  • Current procedures for molecular deposition and patterning include drop projection, micro-contact printing, electron- beam lithography, and dip-pen nanolithography. However, these methods are not suitable for depositing single molecules onto the surface. It can be desirable to fabricate an array having single molecules, particular single nucleic acid molecules, deposited at each location. It is also desirable to use a deposition method that is easily adjustable to permit tunable spacing distances between the deposited molecules.
  • the disclosure relates to a composition comprising one or more spacers linked to one or more anchors to form a spacer-anchor complex.
  • the anchor includes a first functional group that binds to, or otherwise mediates the linkage of the anchor to, a suitably modified surface.
  • the anchor can further include a second functional group that binds to, or otherwise mediates the linkage of the anchor to, the spacer.
  • the spacer and/or the anchor can include a polymeric portion comprised of a linear or branched polymer, for example a protein, nucleic acid or carbohydrate polymer.
  • the spacer, the anchor, or both the spacer and anchor can include nucleic acid, which can be single or double stranded.
  • the spacer includes a nucleic acid that is folded into a three-dimensional shape.
  • the spacer comprises a double- stranded nucleic acid molecule that between about 1-1000 kilobase pairs in length, for example between about 50-500 kilobase pairs in length, 75-300 kilobase pairs in length, or 100-200 kilobase pairs in length.
  • the double- stranded nucleic acid molecule is about 50-300 kilobase pairs in length, 50-300 kilobase pairs in length, or 75-250 kilobase pairs in length.
  • the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e
  • the disclosure relates to a system comprising one or more spacer- anchor complexes, where each complex includes: one or more spacers linked to one or more anchors, where at least one spacer includes a polymeric portion and at least one anchor includes a functional group capable of mediating the attachment of the complex to a surface.
  • the anchor can further include a second functional group that links the anchor to the spacer.
  • a single spacer is linked to a single anchor to form the spacer-anchor complex.
  • the anchor includes a nucleic acid molecule that is linked to the polymeric molecule of the spacer.
  • the polymeric molecule includes nucleic acid, for example a double- stranded nucleic acid molecule.
  • the spacer comprises a double-stranded nucleic acid molecule that between about 1-1000 kilobase pairs in length, for example between about 50-500 kilobase pairs in length, 75-300 kilobase pairs in length, or 100-200 kilobase pairs in length.
  • the double- stranded nucleic acid molecule is about 50-300 kilobase pairs in length, 50-300 kilobase pairs in length, or 75-250 kilobase pairs in length.
  • the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e.g., length, width, height and/or diameter).
  • the disclosure relates to methods for preparing an array of molecules, comprising: contacting a solid or semi-solid surface with at least one spacer-anchor complex, where the at least one spacer-anchor complex includes one or more polymeric spacers linked to an anchor, under conditions where the anchor of the spacer-anchor complex binds to the surface.
  • the array is prepared by depositing a population of spacer-anchor complexes onto the surface.
  • the spacer anchor-complex comprises a single spacer linked to a single anchor.
  • the spacer and/or the anchor comprises nucleic acid.
  • each location on the solid surface is deposited with a single spacer-anchor complex.
  • the size-dimension of the spacer typically restricts the spacing between the deposited complexes.
  • the spacing between the deposited complexes of the array can be at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm.
  • the length of the nucleic acid molecule that comprises the spacer can be adjusted to be longer or shorter, in order to tune the size of the three- dimensional spacer, which can be at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension.
  • the spacer can be cleaved from the anchor to reveal an array of deposited, single anchors comprising nucleic acid.
  • These surface-attached anchors can optionally be used to bind/link other biological molecules or non-biological compounds to the surface to create a nanoscale array for analytical procedures.
  • the anchor includes a first functional group
  • the surface includes a second functional group
  • the anchor is linked to the surface via interaction, bonding or association between the first and second functional groups.
  • the polymeric spacer includes a - -
  • the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e.g., length, width, height and/or diameter).
  • the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm,
  • the disclosure relates to methods for preparing an array of molecules, comprising: (a) contacting a solid or semi-solid surface with an anchor including a nucleic acid molecule having a first linking group, where the anchor is linked to a spacer that includes a nucleic acid molecule and where the surface includes a second linking group, under conditions where the first and second linking groups form a bond, thereby linking the anchor to the surface.
  • the contacting is in the presence of a reagent that promotes bond formation between the first and second linking groups.
  • the anchor molecule further comprises a cleavable linker moiety, a binding partner moiety, a reporter moiety, and/or a flexible or rigid moiety.
  • FIG. 1 illustrates an exemplary method for preparing a spacer-anchor complex.
  • FIG. 2 illustrates an exemplary method for preparing a nucleic acid array using the spacer-anchor complexes.
  • FIG. 3 shows microscope images of spacer-anchor complexes.
  • FIG. 3(A) shows spacer- anchor complexes stained with SYBR Green dye.
  • FIG. 3(B) shows spacer-anchor complexes attached to a quantum dot.
  • FIG. 4 shows the nucleotide sequence of coliphage lambda DNA.
  • FIG. 5 illustrates the result of an exemplary assay performed according to Example 6.
  • the disclosure relates to compositions, systems, and methods for preparing surface arrays using spacers.
  • one or more spacers are linked to one or more anchors to form a spacer-anchor complex.
  • the anchor optionally includes at least one functional group capable of linking the anchor to a surface.
  • each spacer-anchor complex includes one or more spacers linked to a single anchor.
  • the biomolecular array is prepared by contacting one or more spacer-anchor complexes with a surface under conditions where the anchor of the spacer-anchor complex binds to or becomes otherwise linked to the surface.
  • the spacer is then cleaved and released, leaving behind the surface-linked anchor.
  • the size or shape of the spacer can optionally be adjusted to modulate the resulting spacing between adjacent surface-linked anchors.
  • this method can be used to produce an array of single anchors, each attached to a discrete location on the surface of the array.
  • the spacing between the surface-linked anchors is sufficient to permit the resolution of individual surface-linked anchors using any suitable detection means of choice.
  • the anchors include functional groups that can serve as points of attachment for biomolecules (e.g., DNA or RNA) or other non- biological molecules of interest.
  • the surface-linked anchors can optionally be used to link these molecules of interest to the surface, thus creating a nanoscale array for analytical procedures.
  • the disclosure relates to a method for forming a molecular array, comprising: contacting one or more spacer-anchor complexes including an anchor linked to a spacer, the spacer including nucleic acid folded to form a three-dimensional shape, with a solid or semi-solid surface under conditions where the anchor of the one or more spacer-anchor complexes binds to the surface, thereby linking the one or more spacer-anchor complexes to the surface.
  • the method further includes cleaving the linkage between the spacer and the anchor to release the spacer from the spacer-anchor complex, thus creating an array of surface- attached anchors.
  • the spacer comprises nucleic acid folded to form a three- dimensional shape.
  • the nucleic acid of the spacer can be double stranded or single stranded.
  • both the spacer and anchor comprise nucleic acid, and the nucleic acid of the anchor can be deposited or otherwise linked to the surface such that single anchors are linked to discrete locations on the surface.
  • the disclosure relates to compositions, systems, and methods for use in preparing arrays of nucleic acid molecules using spacers linked to anchors.
  • the spacer comprises a long, double-stranded, nucleic acid molecule which is folded to form a three- dimensional shaped molecule.
  • the anchor can comprise a short nucleic acid molecule.
  • the arrays can be prepared by delivering the anchors to a solid surface using a spacer which restricts the - -
  • the size of the spacer can be adjusted to facilitate a tunable spacing distance between the anchors.
  • the disclosure relates to spacers that are useful in preparing single molecule arrays for use in a variety of applications.
  • the spacer is linked to the molecules to be arrayed on a suitable surface and is sized so as to achieve the desired distance between adjacent molecules on the array.
  • the spacer comprises one or more linear or branched polymers (e.g., nucleic acid or protein polymers) that are folded to form a three- dimensional shape.
  • the spacer comprises one or more linear, double- stranded nucleic acid molecules that are folded or collapsed to convert the linear form to a 3-dimensional form (Fig. 1).
  • the spacer measures at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in at least one dimension (e.g., length, width, height and/or diameter).
  • a dimension e.g., length, width, height and/or diameter
  • the spacer comprises one or more nucleic acid molecules and the size of the spacer can optionally be adjusted (e.g., tuned) to be larger or smaller in any one or more dimensions (e.g., length, width, depth, and/or diameter), by varying the length of the one or more nucleic acid molecules that comprise the spacer.
  • the one or more nucleic acid molecules can be at least about 500 base pairs in length.
  • the one or more nucleic acid molecules can be at least about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 300, 350, 400, 450, 500, 600, 750, or 1000 kilobase pairs in length, or even longer.
  • the spacer comprises one or more nucleic acid molecules about 3- 200 kilobase pairs in length.
  • the size of the spacer structure can be instrumental in controlling the spacing between adjacent spacer-anchor complexes bound to the surface of an array.
  • the one or more spacer-anchor complexes are bound to the surface at an average distance of between about 400 nm-2000 nm from each other, typically between about 500-1800 nm, even more typically between about 600-1600 nm.
  • the spacer can be linked to an anchor molecule.
  • a single spacer is linked to a single anchor molecule.
  • the nucleic acid molecules can be prepared using recombinant DNA technology, fragmenting larger pieces into shorter pieces, ligating together smaller fragments (including fragments prepared using chemical synthesis methods), phage culture technology, or any combination thereof. Selection of the technique for producing the desired length may depend upon the desired fragment sizes and subsequent preparation steps.
  • the nucleic acid molecule may be nicked, sheared, or treated with an enzyme such as a restriction endonuclease or a nuclease (exonuclease).
  • an enzyme such as a restriction endonuclease or a nuclease (exonuclease).
  • long nucleic acid molecules can be prepared by concatemerization, or ligating shorter pieces together. Any combination of fragmentation or elongation techniques may be practiced in any order to prepare the desired length of nucleic acid molecule.
  • the spacer can include nucleotide sequences that are about 48 kb in length, or about 48.5 kb in length, or about 48,502 bp in length.
  • the spacer can be a double-stranded nucleic acid molecule, comprising nucleotide sequences from any source, including phage, bacterial plasmid, or vector sequences, or fragments thereof.
  • the spacer can include concatemers of phage, plasmid, or vector sequences. The concatemers can be in a head-to-head or head-to-tail arrangement.
  • the spacer can include concatemers of pBR322, or other plasmid sequences.
  • the spacer can include nucleotide sequences which are coliphage, T4, T7, adenovirus, or other viral sequences.
  • the spacer can include double- stranded, linearized sequences from phiX174 virus.
  • the spacer can include the nucleotide sequence from lambda phage genome.
  • the nucleotide sequence and restriction maps of lambda DNA are well known (Fig. 4) (Sanger, et al., 1982 Journal of MoI. Biol. 162:729-773). See for example the complete genomic sequence of the 1-strand of bacteriophage lambda in: GenBank NC_001416.1, GL9626243, and also GenBank J02459.1, Version Jo2459.1, GL215104, Accession Nos. J02459, M17233, M24325, V00636, and X00906.
  • the lambda DNA can be from a commercially-available source (e.g., New England Biolabs, or DL Daniels, et al., 1983, in Appendix II: Complete Annotated Lambda Sequence, eds: RW Hendrix, et al., "Lambda-II", pp. 519-676).
  • a commercially-available source e.g., New England Biolabs, or DL Daniels, et al., 1983, in Appendix II: Complete Annotated Lambda Sequence, eds: RW Hendrix, et al., "Lambda-II", pp. 519-676.
  • Methods for forming three-dimensional structures having desired size dimensions and comprised of linear or branched polymers are also known in the art.
  • large nucleic acid molecules can be folded, condensed or collapsed into three dimensional forms, e.g., toroids, rods, globules, etc, when subjected to suitable conditions, including, for example, condensation increased ionic strength, increased or decreased temperature and/or or exposure to reagents such as - -
  • the nucleic acid molecule that comprises the spacer can be collapsed or folded to form the 3-dimensional form (see Fig. 1) using salt, spermine, spermidine, and/or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the spacer can include wild- type or mutant sequences.
  • the mutant sequences can include nucleotide insertions, deletions, substitutions, or rearrangements.
  • the spacer can include wild-type or mutant coliphage genomic nucleic acid molecules.
  • the spacer can include coliphage genomic nucleic acid molecules, which are isolated from wild-type or mutant host cells.
  • the spacer can include coliphage DNA, e.g., T4 genomic DNA, T7 genomic DNA, lambda genomic DNA, which includes the -indl mutation (C substituted with a T at position 37,589), the -Sam7 mutation (G substituted with an A at position 45,352), -cI857 mutation (C substituted with a T at position 37,742), or the -cI858s7 mutation (G substituted with an A at position 43,082).
  • the spacer can included non-methylated and methylated nucleotides, or other forms of modified nucleotides.
  • the spacer molecule can be isolated from a host strain carrying one or more methylase mutations, such as dcm, dam, or EcoKI methylase.
  • the life cycle of lambda phage, and methods for culturing and producing phage DNA is well known in the art (DL Daniels, et al, 1983 in: "Lambda II" , pgs. 469-517, and pgs. 519-674, eds. RW Hendrix, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor).
  • the spacer can be a nucleic acid molecule having overhang ends, blunt ends, or both.
  • the overhang ends can be generated by restriction enzyme digestion.
  • the overhang ends can include complementary sequences (e.g., cohesive ends) or non-complementary ends.
  • the nucleic acid molecule can be a phage DNA molecule comprising complementary, cohesive ends.
  • the spacer can include complementary cohesive ends that are about 5-20 bases in length.
  • the nucleic acid molecule comprises complementary cohesive ends that are about 12 bases in length.
  • the complementary cohesive ends can include lambda cohesive end sequences, such as: 5' GGGCGGCG ACCT3' and/or 5 ⁇ GGTCGCCGCCC3'.
  • the nucleic acid molecules that comprise the spacers can be prepared using any method, including recombinant DNA technology such as: coliphage DNA preparation, host- amplification of vector DNA; PCR; fragment ligation; fragment scission; and/or linearizing circular molecules; or any combination thereof.
  • recombinant DNA technology such as: coliphage DNA preparation, host- amplification of vector DNA; PCR; fragment ligation; fragment scission; and/or linearizing circular molecules; or any combination thereof.
  • a spacer comprising one or more linear or branched polymer that is folded to form a three-dimensional structure can provide several advantages.
  • One advantage is that the size of the spacer can be adjusted by selecting the length of one or more polymers that comprise the spacer and/or adjusting the reaction conditions employed to fold, condense or otherwise collapse the polymer.
  • Another advantage is that the nucleic acid molecule of the spacer can be bound to the anchor using reaction chemistries that will ensure that only a single anchor is bound to the spacer, thus ensuring that only a single spacer will bind to a given location on the array surface.
  • the disclosure relates to anchors molecules linked to spacer molecules.
  • the anchor molecules can be linear nucleic acid molecules.
  • the anchor molecules can be single- or double-stranded nucleic acid molecules, including DNA, RNA, DNA/RNA hybrids, or analogs thereof.
  • the anchor may not include any nucleic acid but instead may include functional groups that serve as attachment sites for nucleic acids or other molecules.
  • the spacer attached or otherwise linked to the anchor is a "spacer-anchor" complex.
  • the spacer can be used to deliver the anchor to a solid surface to prepare an array of anchor molecules.
  • the size/dimension of the spacers restricts the distance between the anchor molecules which are immobilized to the solid surface (see the scheme in Fig. X).
  • the anchor can be prepared using any procedure, including: recombinant DNA technology and/or chemical synthesis.
  • the length of the anchor can be selected to optimize tethering, proximity, flexibility, rigidity, and/or orientation of the attached spacer.
  • the anchor can be about 5-50, or about 5-40, or about 5-30, or about 5-20, or about 5-10 nucleotides in length. In one embodiment, the anchor can be about 15-20 nucleotides in length.
  • the sequence of the anchor can include any nucleotide sequence, including homo- polymeric or hetero-polymeric sequences.
  • the anchor molecule can be homo- polymeric-A, homo-polymeric -G, homo-polymeric -C, homo-polymeric -T, homo-polymeric -U, or homo-polymeric-I.
  • the anchor can include any restriction enzyme sequence.
  • the anchor can have a hetero-polymeric sequence, such as: 5'- GGGCGGCGACCTGGGT-Biotin-dT-3 ' .
  • the number of spacer and anchor molecules linked to each other can be varied.
  • one anchor can be linked to one spacer, or multiple anchors can be linked to one spacer, or one anchor can be linked to multiple spacers.
  • the spacer and anchor are linked to each other using conventional linking methods, which include: annealing over-hang ends; ligation; linking chemistry; bridge-primer hybridization; click chemistry; and binding partners.
  • the anchor can include a terminal end having a sequence that is complementary to the over-hang end to facilitate annealing the spacer and anchor together.
  • the spacer and anchor can be catalytically linked together using a ligase.
  • the spacer and anchor can be linked together using any type of linker moiety or linking chemistry, including: NHS ester chemistry; click chemistry; or aldehyde/hydrazide chemistry. Other linking chemistry schemes are possible.
  • linker moiety or linking chemistry including: NHS ester chemistry; click chemistry; or aldehyde/hydrazide chemistry.
  • Other linking chemistry schemes are possible.
  • the anchors are adaptable since they can include at least one linker moiety, reporter moiety, binding partner moiety, extension moiety, and/or flexibility/rigidity moiety.
  • the anchor can include at least one suitable linker (e.g., functional groups) that can be cleaved.
  • the anchor can include at least one suitable linker for operable linkage to the spacer, the solid surface, or to other compounds.
  • the anchors can include at least one suitable linker for operable linkage to reporter moieties, cleavable linkers, extension moiety, and/or binding partners.
  • the anchors can include moieties that replace one or more nucleosides and can alter the flexibility or rigidity of the anchor.
  • the rigidity moieties can include polyimide or phenyl units.
  • the anchor can include at least one extension moiety which serves as an extender.
  • the anchors can include at least one suitable linker, binding partner, reporter moiety, extension moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement on the anchors and at any position on the anchors (e.g., 5' or 3' ends, or internal positions).
  • the anchor can include a sequence that is recognized for cleavage by a restriction endonuclease enzyme.
  • the 5' end of the anchors can include an amino, NHS ester, alkyne, or aldehyde functional group that mediate linkage to the spacer or solid surface.
  • the 3' end of the anchors can include an amino or aldehyde functional group for linkage to the spacer or solid surface.
  • the anchor can include polyethylene glycol or polyethylene oxide units, which have a polymer coil volume in aqueous environments that permit the units to extend into the environment rather than curl/coil.
  • the anchor can include about 1-12 PEG or PEO units.
  • the anchor comprising the PEG or PEO units can be prepared by employing phosphoramidite chemistry.
  • an amino-derivatized solid phase can be used to link the PEG or PEO units to the anchor molecule (see the method disclosed by Woo in U.S. Patent No. 5,625,052).
  • successive PEO units can be added to the anchor using a base-modified deoxyuridine phosphoramidite with a TFA-protected amine (e.g., LAN from Molecular Biosystems, as disclosed by Grossman in U.S. Patent No. 5,807,682).
  • the anchor comprising the PEG or PEO units can also include an amine group (e.g., aminohexyl group) at the 5' or 3' end.
  • the 5' or 3' end of the anchor can be linked to the spacer.
  • the 5' or 3' end of the anchor can be linked to the solid surface.
  • the anchor comprising the PEG or PEO units can be prepared starting from the 5' or the 3' end of the nucleic acid molecule using solid phase synthesis methods and/or employing nucleic acid synthesis equipment (e.g., ABI 394 DNA synthesizer).
  • the anchor comprises the sequence: 5'-(ATCG)-S-S-TAT-biotin- (PEO) N -amine -3', where the "ATGC” can include any nucleotide sequence, and the "S-S” can include a thiol linker or a photocleavable linker, and "N" can be about 1-12 PEO units.
  • the anchor includes five PEO units.
  • the anchors comprise at least one suitable linker or chemical bond that attaches the anchor to the: spacers, reporter moieties, binding partners, flexibility/rigidity moieties, solid surfaces, and/or other compounds.
  • the suitable linkers, reporter moieties, binding partners, and flexibility/rigidity moieties do not interfere with the function or activity of the spacer or the anchor, or with each other.
  • the suitable linkers can be selected to optimize proximity, length, distance, orientation, charge, or flexibility or rigidity.
  • the suitable linker can be linked to the spacer, reporter moieties, solid surfaces, and/or other compounds, via covalent bonding, non-covalent bonding, ionic bonding, hydrophobic interactions, or any combination thereof.
  • non-covalent attachment includes: ionic, hydrogen bonding, dipole-dipole interactions, van der Waals interactions, ionic interactions, and hydrophobic interactions.
  • non-covalent attachment includes: nucleic acid hybridization, protein aptamer-target binding, electrostatic interaction, hydrophobic interaction, non-specific adsorption, and solvent evaporation.
  • the suitable linker can include a short or long spacer-arm, a hydrophilic spacer-arm, or an extended spacer-arm.
  • the suitable linker can be rigid or flexible.
  • the suitable linker can be linear, non-linear, branched, bifunctional, trifunctional, homofunctional, or heterofunctional. Many cleavable, and bifunctional (both homo- and hetero-bifunctional) spacer arms with varying lengths are available commercially. Some linkers have pendant side chains or pendant functional groups, or both.
  • the suitable linker can be resistant to heat, salts, acids, bases, light, chemicals, or shearing forces or flow.
  • the suitable linker can include multiple amino acid residues, such as a poly-arginine linker.
  • the suitable linker can be a cleavable, self-cleavable, or fragmentable linker.
  • the linker can be cleavable or fragmentable using temperature, enzymatic activity, chemical agent, and/or electromagnetic radiation.
  • the linker attachment can be reversible.
  • the anchor can include at least one suitable cleavable linker to permit release of the spacer.
  • the suitable cleavage linker can include a disulfide, silyl, amide, thioamide, ester, thioester, vicinal diol, phosphoramidite, or hemiacetal group.
  • Other cleavable bonds include enzymatically-cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).
  • the photo-cleavable linkers include nitrobenzyl derivatives, phenacyl groups, and benzoin esters. Analogs of the 2-nitrobenzyl linker, and other photocleavable linkers including: 2- nitrobenzyloxycarbonyl; nitroveratryl; 1-pyrenylmethyl; 6-nitroveratryloxycarbonyl; dimethyldimethoxybenzyloxyc arbonyl ; 5 -bromo-7 -nitroindolinyl ; O -hydroxy- alpha-methyl- cinnamoyl; methyl-6-nitroveratryloxycarbonyl; methyl-6-nitropiperonyloxycarbonyl; 2- oxymethylene anthraquinone; dimethoxybenzyloxy carbonyl; 5-bromo-7 -nitroindolinyl; O- hydroxy- alpha- methyl cinnamoyl; and 2-oxymethylene anthriquinone (see: McGaIl, U.S.
  • the photocleavable linkers can be illuminated with an electromagnetic source at about 320-800 nm, depending on the particular linker, to achieve cleavage.
  • the self-cleaving linker can be a trimethyl lock or a quinone methide linker.
  • the cleavable linker can be a commercially-available linker. - -
  • the photocleavable linker is a phosphoramidite (e.g., Glen Research, catalog #10-4920-xx).
  • the cleavable disulfide linker is a thiol modifier (e.g., Glen Research, catalog #10-1936-xx).
  • the fragmentable linker can be capable of fragmenting in an electronic cascade self- elimination reaction (Graham, U.S. published patent application No. 2006/0003383; and Lee, U.S. published patent application No. 2008/0050780).
  • the fragmentable linker comprises a trigger moiety.
  • the trigger moiety comprises a substrate that can be cleaved or "activated" by a specified trigger agent. Activation of the trigger moiety initiates a spontaneous rearrangement that results in the fragmentation of the linker and release of the enjoined molecules (e.g., spacer and anchor).
  • the trigger moiety can initiate a ring closure mechanism or elimination reaction.
  • Various elimination reactions include 1,4-, 1,6- and 1,8 -elimination reactions.
  • the trigger moiety can include a cleavage site that is cleavable by a chemical reagent or enzyme.
  • the trigger moiety can include be a cleavage recognition site that is cleavable by a sulfatase (e.g., SO 3 and analogs thereof), esterase, phosphatase, nuclease, glycosidase, lipase, esterase, protease, or catalytic antibody.
  • the suitable linker comprises about 1-100 plural valent atoms.
  • the linker moiety comprises about 1-40 plural valent atoms, or more, selected from the group consisting of C, N, O, S and P.
  • the suitable linker can include any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds or carbon- sulfur bonds.
  • Exemplary linking members include a moiety that includes -C(O)NH-,
  • the linkers can include a combination of moieties selected from amine, alkyl, alkylene, aryl, -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O)-, -S(O) n - , where n is 0, 1, 2, 3, 4, 5, or 6-membered monocyclic rings and optional pendant functional groups, for example sulfo, hydroxy and carboxy.
  • the suitable linker can include a pendant side chain or pendant functional group, or both.
  • pendant moieties include hydrophilicity modifiers, for example solubilizing groups such as sulfo (-SO 3 H- or -SO 3 - ).
  • the trifunctional linker can be linked to multiple reporter moieties (the same or different reporter moieties) for dendritic amplification of the signal emitted by the reporter moieties (Graham, U.S. published patent application Nos. 2006/0003383 and 2007/0009980).
  • the suitable linker can be a rigid linker which can be used, for example, to improve a FRET signal by optimizing the orientation of the energy transfer dye.
  • rigid linkers include benzyl linkers, proline or poly-proline linkers (S. Flemer, et al., 2008 Journal Org. Chem. 73:7593-7602), bis-azide linkers (M.P.L. Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkers synthesized by modifying the so-called "click" chemistry scheme that is described by Megiatto and Schuster (2008 Journal of the Am. Chem. Soc. 130:12872-12873).
  • click chemistry can include azide alkyne Huisgen cycloaddition or alkynyl linkage.
  • the suitable linker can be capable of energy transfer, such as those disclosed by Ju in U.S. published patent application No. 2006/0057565.
  • the anchor can include an NHS ester linkage, such as one provided by an NHS-carboxy-dT compound (e.g., Glen Research, catalog # 10-1535-xx).
  • the anchor can include an aldehyde linkage, such as one provided by a 5- formylindole-CE phosphoramidite (e.g., Glen Research, catalog #10-1934-xx) or a 5'-aldehyde- modifier C2 phosphoramidite compound (e.g., Glen Research, catalog #10-1933-xx).
  • the anchor can include an alkynyl linkage such as one provided by a 5'- hexynyl phosphoramidite compound (e.g., Glen Research, catalog # 10-1908-xx).
  • the alkynyl linkage can be used for click chemistry.
  • the 5' end of the anchor can include an amino group, such as one provided by a 3'-amino-modifier C7 CPG 500 compound (e.g., Glen Research, catalog #20-2957- xx).
  • the 3' end of the anchor can include an amino group, such as one provided by a 5'-amino-modifier C6 compound (e.g., Glen Research, catalog #10-1906-xx).
  • the anchors can include non-natural nucleotides having reactive group that will attach to a surface reactive group.
  • the non-natural nucleotides include peptide nucleic acids, locked nucleic acids, oligonucleotide phosphoramidates, and oligo- - -
  • the anchors can be modified with one or more amino groups at their 5' or 3' ends, or internally, for attachment to modified surfaces.
  • the amino group at the 5' end of the anchors includes: a simple amino group; a short or long tethering arm having one or more terminal amino groups; or an amino-modified thymidine or cytosine.
  • the amino group at the 3' end of the nucleic acid molecule is typically initially protected by a fluorenylmethylcarbamoyl (Fmoc) group.
  • the protecting group can be removed and acylated with an appropriate succinimidyl ester, such as an N-hydroxy succinimidyl ester (NHS ester).
  • the anchors can carry internal amino groups for binding to the solid surface.
  • 2' amino modified nucleic acid molecules can be produce by methoxyoxalamido (MOX) or succinyl (SUC) chemistry to produce nucleotide analogs having amino linkers attached at the 2' C of the sugar moiety.
  • the anchors can include succinylated nucleic acid molecules which can be attached to aminophenyl- or aminopropyl-modified surfaces (B. Joos et al., 1997 Anal. Biochem. 247: 96-101).
  • the anchors can include a thiol group which is placed at the 5' or 3' end of the anchors.
  • the thiol group can form reversible or irreversible disulfide bonds with the surface.
  • the thiol attached to the 5' or 3' end of the anchors can be a phosphoramidate.
  • the phosphoramidate can be attached to the 5' end using S-trityl-6-mercaptohexyl derivatives.
  • the anchors can be reacted with modifying reagents such as: carbodiimides (e.g., dicyclohexylcarbodiimide, DCC), carbonyldiimidazoles (e.g., carbonyldiimidazole, CDI 2 ), or potassium periodate.
  • modifying reagents such as: carbodiimides (e.g., dicyclohexylcarbodiimide, DCC), carbonyldiimidazoles (e.g., carbonyldiimidazole, CDI 2 ), or potassium periodate.
  • the anchors can have protective photoprotective caps (Fodor, U.S. Patent No. 5,510,270) capped with a photoremovable protective group.
  • DMT-protected anchors can be immobilized to the surface via a carboxyl bond to the 3' hydroxyl of the nucleoside moiety (Pease, U.S. Pat. No. 5,599,695; Pease et al., 1994 Proc. Natl. Acad. Sci. USA 91:5022- 5026).
  • the anchors can be functionalized at their 5' ends with activated 1-O-mimethoxytrityl hexyl disulfide l'-[(2-cyanoethyl)-N,N-diisopropyl)] phosphoramidate (Rogers et al., 1999 Anal. Biochem. 266:23). Binding Partner Pairs
  • Binding partner pairs can be used to link the spacer, anchor, reporter moiety, flexibility/rigidity moiety, other compounds, and/or solid surface, to each other in any combination.
  • one member of the binding partner pair can be linked to one end of the anchor, and the other member of the binding partner can be linked to the solid surface.
  • binding partners include: biotin or desthiobiotin or photoactivatable biotin and their binding partners avidin, streptavidin, NEUTRA VIDIN, or CAPTA VIDIN; His-tags which bind with nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni- NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium-calcium binding protein (CBP); acetylcholine and receptor-acetylcholine; protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which binds to antibody or antibody fragments, particularly antigens such as digoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and their respective antibodies; mouse immunoglobulin and goat anti-
  • binding partner pairs include: artificial biotin binding sequences, such as an AVI-TAG (Avidity LLC).
  • the artificial biotin binding sequence comprises a biotin ligase sequence.
  • the biotin binding sequence comprises the sequence (in single-letter amino acid symbols) GLNDIFEAQKIEWHE.
  • the biotin can bind the lysine (K) residue within the artificial biotin binding sequence.
  • the artificial biotin binding sequence can be used for site-specific and/or mono- biotinylation of proteins. See for example Chapmann- Smith and Cronan 1999 Trends Biochem Sci 24:359-363; M.A. Eisenberg, et al., 1982 J. Biol Chem 275:15167-15173; J.E. Cronan 1990 J Biol Chem 265:10327-10333; and PJ. Schatz 1993 Biotechnology 11:1138-1143.
  • Any linking chemistry scheme can be used to generate reactive groups for linking together the spacer, anchor, cleavable linkers, binding partners, reporter moieties, flexibility/rigidity moieties, or other compounds, and/or solid surfaces, in any combination and in - -
  • the reactive groups include: amine, aldehyde, hydroxyl, sulfate, carboxylate groups, and others.
  • Reacting an aldehyde or ketone with a hydrazine to form a hydrazone bond Reacting an aldehyde or ketone with a hydroxylamine to form an oxime bond.
  • Reacting an alkyl halide with an amine or aniline to form an alkyl amine bond Reacting alkyl halides, alkyl sulfonates, diazoalkanes, or epoxides with carboxylic acids to form an ester bond.
  • Reacting an anhydride with an amine or aniline to form a carboxamide or imide bond.
  • Reacting an aryl halide with a thiol to form a thiophenol bond Reacting an aryl halide with an amine to form an aryl amine bond.
  • Reacting an epoxide with a thiol to form a thioether bond Reacting a haloplatinate with an amino or heterocyclic group to form a platinum complex.
  • Reacting a halotriazine with an amine or aniline to form an aminotriazine bond Reacting a halotriazines with an alcohol or phenol to form a triazinyl ether bond. Reacting an imido ester with an amine or aniline to form an amidine bond. Reacting an isocyanate with an amine or aniline to form a urea. Reacting an isocyanate with an alcohol or phenol to form a urethane bond. Reacting an isothiocyanate with an amine or aniline to form a thiourea bond. Reacting a phosphoramidate with an alcohol to form a phosphite ester bond.
  • Reacting a silyl halide with an alcohol to form a silyl ether bond Reacting a sulfonate ester with an amine or aniline to form an alkyl amine bond. Reacting a sulfonyl halide with an amine or aniline to form a sulfonamide bond.
  • the linking chemistry scheme can include "click” chemistry schemes (Gheorghe, et al., 2008 Organic Letters 10:4171-4174).
  • the suitable linking scheme can include reacting the components to be linked in a suitable solvent in which both are soluble.
  • Water-insoluble substances can be chemically modified in an aprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble materials can be accomplished using reactive compounds to make them more readily soluble in organic solvents.
  • Polymers of ethylene oxide can be used to attach the spacer, anchor, cleavable linkers, binding partners, reporter moieties, flexibility/rigidity moieties, or other compounds, and/or solid surfaces, to each other in any combination.
  • polymers of ethylene oxide include: polyethylene glycol (PEG), such as short to very long PEG; branched PEG; amino-PEG-acids; PEG-amines; PEG-hydrazines; PEG-guanidines; PEG-azides; biotin-PEG; PEG-thiols; and PEG- maleinimides.
  • PEG includes: PEG-1000, PEG-2000, PEG-12-OMe, PEG- 8-OH, PEG-12-COOH, and PEG- 12-NH 2 .
  • the anchor can be immobilized to the solid surface via hybridization to a capture probe (e.g., nucleic acid probe) which is linked to the solid surface.
  • a capture probe e.g., nucleic acid probe
  • the surface may comprise capture nucleic acid probes that form complexes with the anchors.
  • the 5' or 3' end of the anchor can hybridize to the capture probe.
  • the capture probes can include oligonucleotide clamps (U.S. Pat. No. 5,473,060).
  • the parameters for selecting the length and sequence of the capture probes are well known (Wetmur 1991 Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259; Britten and Davidson, chapter 1 in: Nucleic Acid Hybridization: A Practical Approach, Hames et al, editors, IRL Press, Oxford, 1985).
  • the length and sequence of the capture probes may be selected for sufficiently stability during low and/or high stringency wash steps.
  • the length of the capture probes ranges from about 6 to 50 nucleotides, or from about 10 to 24 nucleotides, or longer.
  • the capture probes can be immobilized to the surface via a single or multiple biotin/avidin interactions.
  • a dual anchor can be used to immobilize the capture probe and anchor to the solid surface.
  • the 5' or 3' end of the anchor or capture probe can be linked to a biotin molecule.
  • the solid surface can be linked to avidin-liked molecules (e.g., avidin).
  • the avidin molecules are capable of binding up to four biotin molecules, permitting stable binding of a biotin end-labeled duplex (e.g., capture probe/anchor) (Buzby, U.S. Patent No. 7,220,549). Linking Groups on Solid Surfaces
  • the surface can be modified to bind amino-modified anchors.
  • 5' amino-modified nucleic acid molecules can be attached to surfaces modified with silane, such as epoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res. 22:2121- 2125; W. G. Beattie et al., 1995 MoI. Biotechnol. 4:213-225) or isothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465).
  • silane such as epoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res. 22:2121- 2125; W. G. Beattie et al., 1995 MoI. Biotechnol. 4:213-225) or isothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465).
  • Acylating reagents
  • the acylating reagents include: isothiocyanates, succinimidyl ester, and sulfonyl chloride.
  • the amino-modified anchors can attach to surface amino groups which have been converted to amino reactive phenylisothiocyanate groups by treating the surface with p-phenylene 1,4 diisothiocyanate (PDC).
  • PDC p-phenylene 1,4 diisothiocyanate
  • the surface amino groups can be reacted with homobifunctional crosslinking agents, such as disuccinimidylcaronate (DCS), disuccinimidyloxalate (DSO), phenylenediisothiocyanate (PDITC) or dimethylsuberimidate (DMS) for attachment to the amino-modified nucleic acid molecules.
  • DCS disuccinimidylcaronate
  • DSO disuccinimidyloxalate
  • PDITC phenylenediisothiocyanate
  • DMS
  • metal and metal oxide surfaces can be modified with an alkoxysilane, such as 3- aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GOPMS).
  • APTES 3- aminopropyltriethoxysilane
  • GPMS glycidoxypropyltrimethoxysilane
  • the surface can be treated with an alkylating agent such as iodoacetamide or maleimide for linking with thiol-modified nucleic acid molecules.
  • silane-treated surfaces e.g., glass
  • SMPB succinimidyl 4-(malemidophenyl)butyrate
  • thiol-modified surfaces can be used to attach anchors carrying disulfide groups (Y. H. Rogers et al., 1999 Anal. Biochem. 266:23-30).
  • the surface can be coated with a polyelectrolyte multilayer (PEM) via light-directed attachment (U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837) or via chemical attachment.
  • PEM chemical attachment can occur by sequential addition of polycations and polyanions (Decher, et al., 1992 Thin Solid Films 210:831-835).
  • the glass surface can be coated with a polyelectrolyte multilayer which terminated with polyanions or polycations.
  • the polyelectrolyte multilayer can be coated with biotin and an avidin-like compound.
  • Biotinylated molecules can be attached to the PEM/biotin/avidin coated surface (Quake, U.S. Patent Nos. 6,818,395, 6,911,345, and 7,501,245).
  • the surface is coated with a compound that increases electrostatic interaction between the surface and nucleic acid molecules (e.g., anchors and capture probes).
  • the surface can be coated with poly-D-lysine or 3-aminopropyltriethoxysilane (Schwartz, U.S. Patent Nos. 6,221,592, 6,294,136; and Schwartz, U.S. published patent application Nos. 2006/275806 and 2007/0161028).
  • the surface can be coated with one or more linking agents, including: symmetrical bifunctional reagents, such as bis succinimide (e.g., bis-N-hydroxy succinimide) and maleimide (bis-N-hydroxy maleimide) esters, or toluene diisocyanate.
  • the linking agents can be heterobifunctional cross-linkers including: m-maleimido benzoyl-N-hydroxy succinimidyl ester (MBS); succinimidyl-4-(p-maleimido phenyl)-Butyrate (SMPB); and succinimidyl-4-(N- Maleimidomethyl)Cyclohexane-l-Carboxylate (SMCC) (L. A. Chrisey et al., 1996 Nucleic Acids Res. 24:3031-3039).
  • MBS m-maleimido benzoyl-N-hydroxy succinimidyl ester
  • SMPB succinimidyl-4-(p-maleimid
  • a glass surface can be layered with a gold (e.g., about 2 nm layer) which is reacted with mercaptohexanoic acid.
  • the mercaptohexanoic acid can be placed in a patterned array.
  • the mercaptohexanoic acid can be reacted with PEG.
  • the PEG can be reacted to bind the anchors. Any of these procedures can be used to link the solid surface to the anchors (or capture probes).
  • the anchor can be linked to at least one reporter moiety.
  • the reporter moiety generates, or causes to generate, a detectable signal.
  • the reporter moiety can be used to locate the anchor (e.g., locate the immobilized anchor on the solid surface).
  • Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent (including energy transfer), phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme.
  • the reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events).
  • a proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other.
  • the reporter moieties may be selected so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction.
  • Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles.
  • the signals from the different reporter moieties do not significantly overlap or interfere, by quenching, colorimetric interference, or spectral interference.
  • the chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, or p-nitrophenyl phosphate, and derivatives thereof.
  • the chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetane compound.
  • the 1,2-dioxetane compound includes disodium 2-chloro-5-(4-methoxyspiro[l,2-dioxetane-3,2'- (5-chloro-)tricyclo[3,3,l-l 3 ' 7 ]-decan]-l-yl)-l -phenyl phosphate (e.g., CDP-STAR) , chloroadamant-2'-ylidenemethoxyphenoxy phosphorylated dioxetane (e.g., CSPD) , and 3-(T- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (e.g., AMPPD).
  • the fluorescent moiety includes: rhodols; resorufins; coumarins; xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins; phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles; stilbenes; squarenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY- 630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678,
  • the fluorescent moiety can be a quencher dye, including: ATTO 540Q, ATTO 580Q, and ATTO 612Q (Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35 (Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (Glen Research).
  • the fluorescent moiety can be a 7-hydroxycoumarin-hemicyanine hybrid molecule which is a far- red emitting dye (Richard 2008 Org. Lett. 10:4175-4178).
  • the fluorescent moiety may be a fluorescence-emitting metal such as a lanthanide complex, including those of Europium and Terbium.
  • the anchor can be linked to an energy transfer donor and/or acceptor moiety.
  • the energy transfer donor can be a nanocrystal (e.g., quantum dot) or fluorescent dye.
  • the energy transfer acceptor moiety can be a fluorescent dye.
  • the energy transfer donor is capable of absorbing electromagnetic energy (e.g., light) at a first wavelength and emitting excitation energy in response.
  • the energy acceptor is capable of absorbing excitation energy emitted by the donor and fluorescing at a second wavelength in response.
  • the donor and acceptor moieties can interact with each other physically or optically in a manner that produces a detectable signal when the two moieties are in proximity with each other.
  • a proximity event includes two different moieties (e.g., energy transfer donor and acceptor) approaching each other, or associating with each other, or binding each other.
  • moieties e.g., energy transfer donor and acceptor
  • the donor and acceptor moieties can transfer energy in various modes, including: fluorescence resonance energy transfer (FRET) (L. Siryer j 97S Ann, Rev. Bioehem. 47: S 19-
  • FRET fluorescence resonance energy transfer
  • the energy transfer pair can be FRET donor and acceptor moieties.
  • FRET is a distance-dependent radiation less transmission of excitation energy from a donor moiety to an acceptor moiety.
  • the efficiency of FRET energy transmission is dependent on the inverse sixth-power of the separation distance between the donor and acceptor, which is approximately 10- 100 Angstroms.
  • FRET is useful for investigating changes in proximity between and/or within biological molecules.
  • FRET efficiency may depend on donor-acceptor distance r as 1/r 6 .
  • the distance where FRET efficiency is 50% is termed R 0 , also known as the Forster distance.
  • R 0 is unique for each donor-acceptor combination and may be about 5 to 10 nm.
  • the efficiency of FRET energy transfer can sometimes be dependent on energy transfer from a point to a plane which varies by the fourth power of distance separation (E, Jares- Erijrnan, ⁇ t a]., 2003 N ⁇ at BiotechnoL 21:1387 ).
  • the disclosed embodiments relate to spacer-anchor complexes comprising spacers linked to anchors.
  • one spacer is linked to one anchor.
  • the anchor can include at least one suitable cleavable moiety, binding partner, reporter moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement on the adaptors.
  • the 5' or 3' end of the anchors can include a suitable linking moiety for attachment to the solid surface.
  • the 5' or 3' end of the anchors can be linked to the solid surface.
  • the spacer- anchor complexes can include (listed in the 5' to 3' order): a reporter moiety, a cleavable moiety, a binding partner, and a 3' amino or aldehyde group.
  • the spacer- anchor complexes can include (listed in the 5' to 3' order): a 5' functional group (e.g., amino, NHS ester, alkyne, or aldehyde), binding partner, cleavable moiety, and reporter moiety.
  • a 5' functional group e.g., amino, NHS ester, alkyne, or aldehyde
  • surfaces e.g., solid surfaces
  • the immobilized anchors may be attached to the surface at their 5' ends or 3' ends, along their length, or along their length with a 5' or 3' portion exposed.
  • the immobilized anchors may be attached to the surface in a manner that renders them resistant to removal or degradation, including procedures that involve washing, flowing, temperature or pH changes, and reagent changes.
  • the anchors may be reversibly attached to the surface.
  • the surface may be a solid surface, and includes planar surfaces, as well as concave, convex, or any combination thereof.
  • the surface may comprise texture (e.g., etched, cavitated or bumps).
  • the surface includes a nanoscale device, a channel, a well, bead, particle, sphere, filter, gel, or the inner walls of a capillary.
  • the surface can be optically transparent, minimally reflective, minimally absorptive, or exhibit low fluorescence.
  • the surface may be non-porous.
  • the surface may be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic (e.g., polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).
  • plastic e.g., polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).
  • the disclosure generally relates to arrays of immobilized anchors, which are prepared by employing spacers to deliver the anchors to the solid surface.
  • Multiple spacer-anchor complexes are used to deliver multiple anchors (nucleic acid molecules) to the surface.
  • Each spacer-anchor complex delivers a single nucleic acid molecule to a site/location on the surface.
  • the arrangement and space-distance between the anchors is facilitated by the size dimensions of the spacer.
  • the distances between the rows or columns (e.g., pitch) of immobilized anchors can be adjusted by using larger or smaller spacers.
  • spacers having dimensions of about 1-5 micron diameter can be used to deliver the anchors to the solid surface.
  • the immobilized anchors may be arranged in a random or ordered array on a surface.
  • the ordered array includes rectilinear and hexagonal patterns.
  • the surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface to create a patterned array.
  • the anchors can be linked to the regions of the patterned array that include a functional linking moiety that bind to a functional linking moiety on the anchor.
  • multiple anchors can be immobilized onto the surface.
  • Each of the multiple immobilized anchors can bind, capture, or react with at least one biomolecule (e.g., nucleic acid molecules, polypeptides, carbohydrates, lipids, or reagents or compounds). Accordingly, an array of biomolecules can be prepared by binding, capturing, or reacting, the biomolecules with the multiple immobilized anchors.
  • the anchors can each include a cleavable linker, which can be cleaved to release the spacers from the anchors.
  • the released spacers can reveal an array of immobilized nucleic acid molecules.
  • the immobilized anchors can be used to bind the biomolecules or nanocrystals (e.g., quantum dots).
  • the immobilized anchors are single-stranded nucleic acid molecules that can be used to capture (i.e., via hybridization) nucleic acid molecules, such as target molecules for sequencing (e.g., single molecule sequencing).
  • the immobilized anchors are nucleic acid molecules (single- or double- stranded) that can be used to bind to other biological molecules (e.g., polypeptides or antibodies), or bind chemical compounds, or bind drugs (e.g., candidate drugs), or bind to non-biological compounds.
  • other biological molecules e.g., polypeptides or antibodies
  • drugs e.g., candidate drugs
  • the anchors can be immobilized onto a surface having microscopic features that permit manipulation and/or analysis, of biological molecules at a nanoscale level.
  • the microscopic features can be at the micro meter size level, nano meter size level, or pico meter size level, or smaller sized levels.
  • the microscopic features can be prepared from organic and/or inorganic compounds.
  • the microscopic features includes: channels, slits, cavities, pores, pillars, or loops.
  • the microscopic features can have length, width, and height dimensions.
  • the microscopic features can be linear or branched shaped, and/or be attached to inlet and/or outlet ports.
  • the branched microscopic features can form a T or Y junction, or other shape and geometries.
  • the microscopic features can be used for delivering, binding, holding, confining, sorting, separating, enriching, mixing, reacting, streaming, flowing, washing, flushing, elongating, stretching, flushing, or washing the spacers or anchors, or reagents that react with the spacers or anchors.
  • the surface can include one or a plurality of microscopic features, typically more than 5,
  • the dimensions of the microscopic features can be about 1 micron, or about 0.1 micron, or about 0.01 micron, or about 0.001 micron, or about 0.0001 micron.
  • the dimensions of the microscopic features can be between about 10-25 nm, or about 25-
  • the microscopic features can have a trench width equal to or less than about
  • the microscopic features can have a trench depth equal to or less than about 200 nanometers.
  • the surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface in any order.
  • the adhesive layer can bind/link the anchors.
  • the resist layer may not bind/link, or exhibits decreased binding/linking, to the anchors.
  • the microscopic features may be prepared/fabricated from any suitable organic or inorganic compound including: amine, silane, biotin, avidin (or avidin-like compounds), PEG, protein binding partners, silicon, carbon, glass, polymer (e.g., poly-dimethylsiloxane), metals, titanium, aluminum, gold, chromium, platinum, silver, nitrides (e.g., boron nitrides), chromium, gold, synthetic vesicles, silicone, or any combination thereof.
  • suitable organic or inorganic compound including: amine, silane, biotin, avidin (or avidin-like compounds), PEG, protein binding partners, silicon, carbon, glass, polymer (e.g., poly-dimethylsiloxane), metals, titanium, aluminum, gold, chromium, platinum, silver, nitrides (e.g., boron nitrides), chromium, gold, synthetic vesicles, silicone, or any combination thereof.
  • the arrays and/or microscopic features may be prepared/fabricated using any suitable method, including: lithography; photolithography; deep UV lithography; soft lithography; diffraction gradient lithography (DGL); nanoimprint lithography (NIL); interference lithography; contact nanoprinting; self-assembled copolymer pattern transfer; spin coating; electron beam lithography; focused ion beam milling; plasma-enhanced chemical vapor deposition; electron beam evaporation; sputter deposition; bulk or surface micromachining; replication techniques such as embossing, printing, casting and injection molding; etching including nuclear track, chemical, or physical etching, reactive ion-etching, wet-etching; sacrificial layer etching; wafer bonding; channel sealing; and combinations thereof.
  • microscopic features can be prepared on a surface by: applying a photoresist compound to a glass surface; passivating the glass surface with a metal (e.g., aluminum or titanium) using a metal evaporation procedure; and removing the photo-resist to produce a metal- passivated glass surface having islands of glass that can be functionalized for binding the spacer- anchor complexes.
  • a photoresist compound e.g., aluminum or titanium
  • the glass islands can be functionalized with PEG, amines, biotin, and/or avidin-like compounds, to bind the spacer- anchor complexes.
  • the methods for applying and removing resists, metal-passivation on a glass surface, and chemical functionalization, are well known in the art.
  • the solid surface can be coupled to a light source, detector (e.g., photon detector), camera, and/or various plumbing components such as microvalves, micropumps, connecting channels, and microreservoirs for controlled flow (in and/or out) of reagents.
  • the nanoscale device includes: a flow cell; reservoirs for holding reagents; inlet ports in fluid communication with the reservoirs and flow cell for delivering the various reagents; outlet ports in fluid communication with the flow cell; photon detectors; and cameras for determining the location of a signal.
  • the surface of the flow cell can be coated with PEM/biotin/avidin (U.S. Patent Nos. Quake, U.S. Patent Nos.
  • the reagents can be pulled through the inlet or outlet ports via capillary action, or by vacuum (Lawson, U.S. published patent application No. 2008/0219890; and Harris, et al., 2008 Science 320:106-109, and Supplemental Materials and Methods from the supporting online material), or moved via a pressure-driven fluidics system.
  • the reagents can be pulled through the inlet or outlet ports using a passive vacuum source (Ulmer, U.S. patent No. 7,276,720).
  • the flow cell can be a two-sided multi-channel flow cell comprising multiple independently-addressable sample channels and removable loading blocks for sample loading (Lawson, U.S. published patent application No. 2008/0219888).
  • the solid surface can be enclosed by being surmounted with a sealing material using suitable methods. See, for example, U.S. Publication No. 2004/0197843.
  • the surface can include a sample reservoir capable of releasing a fluid, and a waste reservoir capable of receiving a fluid, wherein both reservoirs are in fluid communication with a common reaction area.
  • the surface can include a microfluidic area located adjacent to the nanofluidic area, and a gradient interface between the microfluidic and nanofluidic area that reduces the local entropic barrier for entry into a microscopic feature area (e.g., channels).
  • a microscopic feature area e.g., channels.
  • the solid surface e.g., glass
  • the solid surface can be patterned using photoresists, and/or photolithography or electron-beam lithography.
  • the patterned surface can be passivated by metal evaporation procedures (e.g., aluminum).
  • the photo-resists can be removed.
  • the exposed glass can be functionalized with biotin or amine groups, and the non-functionalized areas can be coated with PEG.
  • the disclosed embodiments generally relate to methods for: binding a nucleic acid molecule to a solid support; binding multiple nucleic acid molecules to a solid support; and preparing a nucleic acid molecule array.
  • the methods can be practiced using the spacer-anchor complexes described herein.
  • the solid surface is contacted with the spacer-anchor complex to bind the anchor to the solid surface.
  • the solid surface can have a random or organized pattern of linking groups to bind to the anchor.
  • the methods can be practiced using suitable conditions that permit binding of the anchor to the solid support, including parameters such as: time, temperature, pH, buffers, reagents, salts, and concentrations of the spacer-anchor complexes.
  • the spacer-anchor complex can be contacted with the solid support for a time that is sufficient to permit binding the anchor portion to the solid support, such as about 10 minutes to 48 hours.
  • the spacer-anchor complex can be contacted with the solid support at a temperature that will permit binding the anchor portion to the solid support, such as about 4 - 50
  • the spacer-anchor complex can be contacted with the solid support at a pH that will permit binding the anchor portion to the solid support, such as about ph 4-12.
  • the suitable pH will be dependent upon the type of linking chemistry between the anchor molecule and solid surface.
  • the buffer or reagents can include a source of monovalent or divalent ions.
  • the buffer can include chelating agents such as EDTA and EGTA, and the like. Binding a Nucleic Acid Molecule to a Solid Surface
  • methods for binding a nucleic acid molecule to a solid surface comprises the steps of: (a) contacting a solid surface with an anchor which comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double- stranded nucleic acid molecule which is 3-170 kilobases in length and which is folded to form a three-dimensional shape, wherein the solid surface comprises a second linking group which can form a bond with the first linking group; and (b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
  • methods for binding multiple nucleic acid molecules to a solid surface comprise the steps of: (a) contacting a solid surface with multiple anchors where each anchor comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double- stranded nucleic acid molecule which is 3-170 kilobases in length and which is folded to form a three-dimensional shape, wherein the solid surface comprises at least one second linking group which can form a bond with the first linking group; and (b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
  • the solid surface comprises a second linking group which is an amino group, aldehyde group, NHS-ester group, alkyne group, or one member of a binding partner pair. In another embodiment, the solid surface comprises a second linking group which is arranged on the surface as a random or organized pattern (e.g. array).
  • the nucleic acid molecule i.e., anchor
  • the nucleic acid molecule is about 5-50 nt or bp in length.
  • the nucleic acid molecule further comprises a cleavable linker moiety, one member of a binding partner pair, a reporter moiety, and/or flexibility/rigidity moiety.
  • the cleavable linker moiety is a photocleavable linker moiety or a chemical-cleavable linker moiety.
  • the binding partner is a biotin molecule.
  • the reporter moiety is a fluorescent dye.
  • the first linking group is an amine, aldehyde, NHS -ester, alkyne, or one member of a binding partner pair.
  • the cleavable linker moiety can be cleaved, for example after the nucleic acid molecule (i.e., anchor) binds to the solid surface. Cleaving the linker moiety and removing the spacer can reveal the single- stranded nucleic acid molecule, which is operable linked to the solid surface.
  • the binding partner can be contacted with the other member of the binding partner pair before, during or after the nucleic acid molecule binds to the solid surface.
  • the fluorescent dye can be excited with an electromagnetic excitation source before, during or after the nucleic acid molecule binds to the solid surface.
  • the double-stranded nucleic acid molecule i.e., spacer
  • the double-stranded nucleic acid molecule comprises a coliphage DNA sequence.
  • the double- stranded nucleic acid molecule comprises a lambda DNA sequence.
  • the double-strand nucleic acid molecule that comprises the spacer can be decreased or increased in length to adjust the size of the three-dimensional shaped spacer.
  • the solid surface is contacted with a homogeneous or heterogeneous population of spacer- anchor complexes.
  • a population of spacer- anchor complexes is a homogeneous population which comprises one type of nucleic acid molecule (anchor) which are linked to one type of double-stranded nucleic acid molecules (spacer).
  • a population of spacer-anchor complexes is a heterogeneous population which comprises one type of nucleic acid molecule (anchor) which are linked to different types of double-stranded nucleic acid molecules (spacer).
  • a population of spacer- anchor complexes is a heterogeneous population which comprises different types of nucleic acid molecules (anchor) which are linked to one type of double- stranded nucleic acid molecules (spacer).
  • the different types of nucleic acid molecules (anchor) comprise different sequences, lengths, cleavable linker moieties, members of a binding partner pair, reporter moieties, linking groups, and/or flexibility/rigidity moieties.
  • the different types of double-stranded nucleic acid molecules (spacer) comprise different sequences and/or lengths.
  • the solid surface can be contacted with a first population of multiple spacer-anchor complexes (homogenous or heterogeneous), and subsequently contacted with a second population of multiple spacer-anchor complexes (homogeneous or heterogeneous).
  • the solid surface can be contacted repeatedly with homogeneous or heterogeneous populations of spacer-anchor complexes.
  • the surface can include multiple second linking groups which are arranged into a random or organized pattern (array).
  • the surface can include additional linking groups (e.g., 3 rd , 4 th , 5 th linking groups, or more) which differ from the second linking groups.
  • the surface can include two or more different types of linking groups which are arranged into a random or organized pattern.
  • the solid surface is contacted with a homogeneous or heterogeneous population of multiple nucleic acid molecules so that type of linking groups on the nucleic acid molecules bind to their cognate linking group on the solid surface.
  • the solid surface comprises a chemical group that does not bind the first linking group.
  • the method further includes the step of: contacting the solid surface with a reagent that modifies the chemical group that does not bind the first linking group (e.g., masking, blocking, and the like).
  • a reagent that modifies the chemical group that does not bind the first linking group e.g., masking, blocking, and the like.
  • the methods comprise an additional step: washing to remove the unbound nucleic acid molecules, or cleaved spacer molecules, or unbound binding partners, or binding reagents/buffers, or reagents that modify the chemical group that does not bind the first linking group.
  • the methods further comprise an additional step: contacting the anchors with biological molecules (e.g., nucleic acid molecules, polypeptides, or antibodies) or chemical compounds or drug candidate compounds.
  • biological molecules e.g., nucleic acid molecules, polypeptides, or antibodies
  • chemical compounds or drug candidate compounds e.g., drug candidate compounds
  • the spacer and anchor molecules are nucleic acid molecules, which can be from any source, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, chemically synthesized, or any combination thereof.
  • the nucleic acid molecules can be isolated from any source including from: organisms such as phage, prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses, cells; tissues, body fluids, or synthesized nucleic acid molecules using recombinant DNA technology or chemical synthesis methods.
  • the nucleic acid molecules can be from any commercially-available source.
  • the spacer and anchor molecules are nucleic acid molecules, which comprise naturally- occurring nucleotides or nucleotide analogs, or any combination thereof. Any portion of the nucleic acid molecule can include a base, sugar, and/or phosphate group analog.
  • the spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include a sugar analog, such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol.
  • the sugar moiety can be: ribosyl, 2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'-dideoxyribosyl, 2',3'- didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl, 2'-aminoribosyl, 2'-fluororibosyl, T- mercaptoriboxyl, 2'-alkylthioribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl, 3'-aminoribosyl, 3'- fluororibosyl, 3'-mercaptoriboxyl, 3'-alkylthioribosyl carbocyclic, acyclic, or other modified sugars.
  • the spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include a hetero cyclic base which includes substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, including naturally- occurring, substituted, modified, or engineered variants, or analogs of the same.
  • the base is capable of forming Watson-Crick and/or Hoogstein hydrogen bonds with an appropriately complementary base.
  • Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N 6 - ⁇ 2 -isopentenyladenine (6iA), N 6 - ⁇ 2 -isopentenyl-2-methylthioadenine (2ms6iA), N 6 - methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2- thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6 -methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thy
  • the spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include phosphate group analogs, such as: phosphoramidate; phosphorothioate; phosphorodithioate; O-methylphosphoroamidite linkages; and peptide nucleic acid backbones and linkages.
  • phosphate group analogs such as: phosphoramidate; phosphorothioate; phosphorodithioate; O-methylphosphoroamidite linkages; and peptide nucleic acid backbones and linkages.
  • nucleic acids analogs include those with bicyclic structures including locked nucleic acids; positive backbones; non-ionic backbones; and non-ribose backbones.
  • the ends and/or interior of the anchors or capture probes may be isolated and modified at their ends and/or the interior of the molecules using well known procedures, including: fragmentation, ligation, hybridization, enzymatic and/or chemical modification, conjugation with a reporter moiety, or linking to an energy transfer (donor or acceptor), or any combination of these procedures.
  • the nucleic acid molecules that comprise the spacer or anchor can be fragmented at random or specific sites using any fragmentation procedure.
  • the nucleic acid molecules can be fragmented using mechanical force, including: shear forces (e.g., small orifice or a needle); nebulization (S. Surzycki 1990 in: "The International Conference on the Status and Future of Research on the Human Genome. Human Genome II", San Diego, CA, pp. 51; and S. J. Surzycki, 2000 in: “Basic Methods in Molecular Biology", New York, NY: Springer- Verlag); or sonication.
  • shear forces e.g., small orifice or a needle
  • nebulization S. Surzycki 1990 in: "The International Conference on the Status and Future of Research on the Human Genome. Human Genome II", San Diego, CA, pp. 51
  • S. J. Surzycki 2000 in: “Basic Methods in Molecular Biology", New York, NY: Springer- Verlag
  • the nucleic acid molecules can be chemically fragmented using, for example: acid- catalyzed hydrolysis of the backbone and cleavage with piperidine; internucleosomal DNA fragmentation using a copper (II) complex of 1,10-phenanthroline (o-phenanthroline, OP), CuII(OP) 2 in the presence of ascorbic acid (Shui Ying Tsang 1996 Biochem. Journal 317:13-16).
  • the nucleic acid molecules can be enzymatically fragmented using type I, II or III restriction endonucleases (N.E. Murray 2000 Microbiol. MoI. Biol. Rev. 64: 412-34; A. Pingoud and A.
  • Enzymatic cleavage of DNA may include digestion using various ribo- and deoxyribonucleases or glycosylases.
  • the nucleic acid molecules can be digested with DNase I or II.
  • the nucleic acid fragments can be generated by enzymatically copying an RNA template. Fragments can be generated using processive enzymatic degradation (e.g., Sl nuclease).
  • the enzymatic reactions can be conducted in the presence or absence of salts (e.g., Mg + , Mn + , and/or Ca + ), and the pH and temperature conditions can be varied according to the desired rate of reaction and results, as is well known in the art.
  • salts e.g., Mg + , Mn + , and/or Ca +
  • the 5' or 3' overhang ends of the nucleic acid molecules that comprise the spacer or anchor can be converted to blunt-ends using a "fill-in” procedure (e.g., dNTPS and DNA polymerase, Klenow, or Pfu or T4 polymerase) or using exonuclease procedure to digest away the protruding end.
  • a "fill-in" procedure e.g., dNTPS and DNA polymerase, Klenow, or Pfu or T4 polymerase
  • exonuclease procedure to digest away the protruding end.
  • nucleic acid molecule ends can be ligated to one or more oligonucleotides using
  • the nucleic acid molecules can be hybridized to one or more oligonucleotides.
  • the oligonucleotides can serve as linkers, anchors, bridges, clamps, anchors, or capture oligonucleotides.
  • the oligonucleotides can include sequences which are: enzyme recognition sequences
  • restriction endonuclease recognition sites e.g., restriction endonuclease recognition sites, DNA or RNA polymerase recognition sites
  • hybridization sites e.g., hybridization sites, or can include a detachable portion.
  • the oligonucleotide can linked to a protein-binding molecule such as biotin or streptavidin.
  • the nucleic acid molecules can be methylated, for example, to confer resistance to restriction enzyme digestion (e.g., EcoRI).
  • the nucleic acid molecule ends can be phosphorylated or dephosphorylated.
  • a nick can be introduced into the nucleic acid molecules using, for example DNase I.
  • a pre-designed nick site can be introduced in dsDNA using a double stranded probe, type II restriction enzyme, ligase, and dephosphorylation (Fu Dong-Jing, 1997 Nucleic Acids Research
  • a nick can be repaired using polymerase (e.g., DNA pol I or phi29), ligase (e.g., T4 ligase) and kinase (polynucleotide kinase).
  • polymerase e.g., DNA pol I or phi29
  • ligase e.g., T4 ligase
  • kinase polynucleotide kinase
  • a poly tail can be added to the 3' end of the fragment using terminal transferase (e.g., polyA, polyG, polyC, polyT, or polyU).
  • terminal transferase e.g., polyA, polyG, polyC, polyT, or polyU.
  • the nucleic acid molecule can be modified using bisulfite treatment (e.g., disodium bisulfite) to convert unmethylated cytosines to uracils, which permits detection of methylated cytosines using, for example, methylation specific procedures (e.g., PCR or bisulfite genomic sequencing).
  • bisulfite treatment e.g., disodium bisulfite
  • methylation specific procedures e.g., PCR or bisulfite genomic sequencing.
  • nucleic acid molecules that comprise the spacer or anchor can be size selected, or the desired nucleic acid molecules can separated from undesirable molecules, using any art-known methods, including gel electrophoresis, size exclusion chromatography (e.g., spin columns), sucrose sedimentation, or gradient centrifugation.
  • the nucleic acid molecules that comprise the spacer or anchor can be amplified using methods, including: polymerase chain reaction (PCR); ligation chain reaction, which is sometimes referred to as oligonucleotide ligase amplification (OLA); cycling probe technology (CPT); strand displacement assay (SDA); transcription mediated amplification (TMA); nucleic acid sequence based amplification (NASBA); rolling circle amplification (RCA); and invasive cleavage technology.
  • PCR polymerase chain reaction
  • OVA cycling probe technology
  • SDA strand displacement assay
  • TMA transcription mediated amplification
  • NASBA nucleic acid sequence based amplification
  • RCA rolling circle amplification
  • Undesired compounds can be removed or separated from the desired nucleic acid molecules to facilitate enrichment of the desired molecules (e.g., spacers or anchors).
  • Enrichment methods can be achieved using well known methods, including gel electrophoresis, chromatography, or solid phase immobilization (reversible or non-reversible).
  • AMPURE beads Amcourt
  • the desired nucleic acid molecules can be enriched using a dialysis procedure, which can be conducted by employing a dialysis membrane having a suitable molecular weight cut-off (MWCO) limit, for a sufficient amount of time, and in a suitable exchange buffer.
  • MWCO molecular weight cut-off
  • the nucleic acid molecules can be enriched using dialysis membranes having about 2K, 3.5K, 7K, or 1OK MWCO.
  • the dialysis procedure can be conducted for about 2-48 hours.
  • the exchange buffer can include Tris at a pH range of about pH 6-9.
  • a biotinylated oligonucleotide was used as the anchor.
  • the oligonucleotide was diluted to 1 ⁇ M in a TE buffer (1 rnM Tris, 0.1 rnM EDTA, pH 8).
  • the sequence of the biotinylated oligonucleotide is: [00167] pGGGCGGCGACCTGGGT-Biotin-dT.
  • the spacer DNA was attached to anchor DNA according to the following procedure (see scheme in Fig. 1).
  • Lambda DNA was ligated to the biotinylated oligonucleotide in a 200 ⁇ L reaction volume: 5 ⁇ L of 1 ⁇ M biotinylated oligonucleotide; 20 ⁇ L of 1OX Taq ligase buffer (20 mM Tris-HCl, 25 mM potassium acetate, 10 mM magnesium acetate, 1 mM NAD, 10 mM dithiothreitol, 0.1 % Triton X-100; pH 7.6); 10 ⁇ L Taq ligase (New England Biolabs, #M0208L); 5 ⁇ L of 1 ⁇ g/ ⁇ L lambda DNA (New England Biolabs, # N301 IS); 106 ⁇ L water.
  • Cycle ligation was performed: (step 1) hold at 80 0 C for 5 minutes; (step 2) hold at 55 0 C for 8 hours; (step 3) 25 x cycles at 85 0 C for 30 seconds and 55 0 C for 60 seconds; (step 4) hold at 4 0 C.
  • the un-ligated oligonucleotides were removed by using a dialysis cassette (Slide-a- Lyzer, Pierce, #MWC0 10,000) in 3 exchanges of 500 mL of Tris, pH 8, for 6 hours per exchange.
  • the dialyzed, spacer-anchor ligation product was stored at 4 0 C.
  • the 5' end of the anchors are functionalized for linkage to the surface by reacting with: an NHS-carboxy-dT compound (e.g., Glen Research, catalog # 10-1535-xx) for NHS-ester linkage; a 5-formylindole-CE phosphoramidite (e.g., Glen Research, catalog #10-1934-xx) or a 5'- aldehyde-modifier C2 phosphoramidite compound (e.g., Glen Research, catalog #10-1933-xx) for an aldehyde linkage; a 5'-hexynyl phosphoramidite compound (e.g., Glen Research, catalog # 10- 1908-xx) for an alkynyl linkage (e.g., for click chemistry); or a 3'-amino-modifier C7 CPG 500 compound (e.g., Glen Research, catalog #20-2957-xx) for amine linkage.
  • an NHS-carboxy-dT compound e.g., Glen Research, catalog
  • the 3' end of the anchors are functionalized for linkage to the surface by reacting with: a 5'-amino-modifier C6 compound (e.g., Glen Research, catalog #10-1906-xx) for amine linage.
  • a 5'-amino-modifier C6 compound e.g., Glen Research, catalog #10-1906-xx
  • the anchors are synthesized to include cleavable linkers using standard chemical synthesis procedures.
  • the anchors include a photocleavable linker such as 3-(4,4'- dimethoxytriyl)-l-(2-nitophenyl)-propan-l-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite (e.g., Glen Research, catalog #10-4920-xx).
  • the anchors include a cleavable disulfide linker such as l-O-dimethoxytrityl-hexyl-disulfie,l'- ⁇ (2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite (e.g., Glen Research, catalog #10-1936-xx).
  • a cleavable disulfide linker such as l-O-dimethoxytrityl-hexyl-disulfie,l'- ⁇ (2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite (e.g., Glen Research, catalog #10-1936-xx).
  • the glass slide (with or without metal-patterning) is oxygen plasma cleaned (300 watts, 5 minutes, 150 mtorr O 2 ) before coating with aminopropyltrimethoxysilane in a YES-1224P chemical vapor deposition chamber.
  • the amine functionalized slide can be stored in a dessicator until reacting with the NHS ester functionalized spacer-anchor complexes.
  • a titanium-patterned slide For heterogeneous modification of a titanium-patterned slide (or other transition metal). Plasma clean the titanium-patterned slide (300 watts, 5 minutes, 150 mtorr O 2 ). Soak the patterned slide overnight in a solution of 5 mM poly(ethylene glycol) phosphonic acid in ethanol. Remove the patterned slide, rinse with copious amounts of ethanol and deionized water, and dry under vacuum. Functionalize the exposed glass surface by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate at 50 0 C overnight. Remove the patterned slide, rinse with copious amounts of deionized water, and dry under vacuum.
  • the slide is washed using 100 mM NaOAc, pH 5.5, four times. Add to the chamber a blocking reagent, 0.2% mPEG-12-amine (QuantaBioDesign, PN 10288), in 100 mM NaOAc, pH 5.5. Incubate for 30 minutes at room temperature. Wash the slide four times with 1 M Tris, pH 7.4. Incubate slide for 5 minutes.
  • a blocking reagent 0.2% mPEG-12-amine (QuantaBioDesign, PN 10288)
  • PEG polyethylene glycol
  • Biotin-TEG l-Dimethoxytrityloxy-3-O-(N-biotinyl-3-aminopropyl)- triethyleneglycolyl-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (from Glen
  • the adaptor was ligated to Lambda DNA as follows: 5 uL of 1 ⁇ M adaptor (in 1 mM
  • Tris and 0.1 mM EDTA, pH 8.0 was mixed with 20 ⁇ L of 1OX Taq ligase buffer (200 mM Tris-
  • Triton X-100 pH 7.6
  • 10 ⁇ L of Taq ligase New England Biolabs
  • 5 ⁇ L Lambda DNA New England Biolabs
  • reaction mixture having a total volume of 200 ⁇ L.
  • cycle ligation including the following steps:
  • Step 1 80 0 C for 5 minutes.
  • Step 2 55°C for 8 hours
  • Step 3 85°C for 30 seconds, followed by 55°C for 60 seconds (repeat for 25 cycles)

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Abstract

The disclosure relates generally to compositions, systems, and methods for use in preparing single molecule arrays, for example, arrays of single nucleic acid molecules, as well as arrays formed using such compositions, systems and methods by using a spacer which restricts the spacing between adjacent components of the array. In an exemplary embodiment, the array is formed by contacting a surface with a spacer-anchor complex including one or more spacers linked to an anchor, wherein the spacer comprises a linear or branched polymeric molecule folded to form a three-dimensional shape of desired size dimensions. The size of the folded spacer may optionally be adjusted to modulate the spacing distance between adjacent array components.

Description

MOLECULAR ARRAYS
[0001] This patent application claims priority to U.S. provisional application no. 61/220,174, filed June 24, 2009, which is hereby incorporated by reference in its entirety. [0002] Throughout this application various patent and non-patent references are cited. The disclosures of these references are hereby incorporated by reference in their entireties into this application for background purposes and to more fully describe the state of the art to which this invention pertains.
FIELD
[0003] The disclosed embodiments are generally directed to compositions, systems and methods for producing arrays of single molecules immobilized at discrete locations on a solid surface. Optionally the spacing between adjacent immobilized molecules is tunable and can be adjusted. In some embodiments, the disclosure relates to arrays of nucleic acid molecules which are immobilized at locations on a solid surface, which can optionally have tunable spacing distances between the nucleic acid molecules, as well as to methods, compositions and systems for preparing such arrays.
BACKGROUND
[0004] Single molecule arrays are used routinely for a wide range of laboratory procedures for diagnostic, screening, and characterization analyses, including polymorphism analyses, elucidation of protein/nucleic acid interactions, screening of candidate pharmaceutical compounds, and sequencing. Preparing such arrays typically involves depositing single molecules onto a solid or semi-solid surface in a random, semi-ordered or ordered pattern. Current procedures for molecular deposition and patterning include drop projection, micro-contact printing, electron- beam lithography, and dip-pen nanolithography. However, these methods are not suitable for depositing single molecules onto the surface. It can be desirable to fabricate an array having single molecules, particular single nucleic acid molecules, deposited at each location. It is also desirable to use a deposition method that is easily adjustable to permit tunable spacing distances between the deposited molecules. SUMMARY
[0005] In some embodiments, the disclosure relates to a composition comprising one or more spacers linked to one or more anchors to form a spacer-anchor complex. In some embodiments, the anchor includes a first functional group that binds to, or otherwise mediates the linkage of the anchor to, a suitably modified surface. Optionally, the anchor can further include a second functional group that binds to, or otherwise mediates the linkage of the anchor to, the spacer. [0006] Typically, the spacer and/or the anchor can include a polymeric portion comprised of a linear or branched polymer, for example a protein, nucleic acid or carbohydrate polymer. In some embodiments, the spacer, the anchor, or both the spacer and anchor can include nucleic acid, which can be single or double stranded. Optionally, the spacer includes a nucleic acid that is folded into a three-dimensional shape. In some embodiments, the spacer comprises a double- stranded nucleic acid molecule that between about 1-1000 kilobase pairs in length, for example between about 50-500 kilobase pairs in length, 75-300 kilobase pairs in length, or 100-200 kilobase pairs in length. In some embodiments, the double- stranded nucleic acid molecule is about 50-300 kilobase pairs in length, 50-300 kilobase pairs in length, or 75-250 kilobase pairs in length. In some embodiments, the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e.g., length, width, height and/or diameter). [0007] In some embodiments, the disclosure relates to a system comprising one or more spacer- anchor complexes, where each complex includes: one or more spacers linked to one or more anchors, where at least one spacer includes a polymeric portion and at least one anchor includes a functional group capable of mediating the attachment of the complex to a surface. Optionally, the anchor can further include a second functional group that links the anchor to the spacer. In a typical embodiment, a single spacer is linked to a single anchor to form the spacer-anchor complex. In some embodiments, the anchor includes a nucleic acid molecule that is linked to the polymeric molecule of the spacer. In a typical embodiment, the polymeric molecule includes nucleic acid, for example a double- stranded nucleic acid molecule. In some embodiments, the spacer comprises a double-stranded nucleic acid molecule that between about 1-1000 kilobase pairs in length, for example between about 50-500 kilobase pairs in length, 75-300 kilobase pairs in length, or 100-200 kilobase pairs in length. In some embodiments, the double- stranded nucleic acid molecule is about 50-300 kilobase pairs in length, 50-300 kilobase pairs in length, or 75-250 kilobase pairs in length. In some embodiments, the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e.g., length, width, height and/or diameter). [0008] In some embodiments, the disclosure relates to methods for preparing an array of molecules, comprising: contacting a solid or semi-solid surface with at least one spacer-anchor complex, where the at least one spacer-anchor complex includes one or more polymeric spacers linked to an anchor, under conditions where the anchor of the spacer-anchor complex binds to the surface. In some embodiments, the array is prepared by depositing a population of spacer-anchor complexes onto the surface. For example, in some embodiments, the spacer anchor-complex comprises a single spacer linked to a single anchor. In some embodiments, the spacer and/or the anchor comprises nucleic acid. Typically, each location on the solid surface is deposited with a single spacer-anchor complex. The size-dimension of the spacer typically restricts the spacing between the deposited complexes. For example, the spacing between the deposited complexes of the array can be at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm. The length of the nucleic acid molecule that comprises the spacer can be adjusted to be longer or shorter, in order to tune the size of the three- dimensional spacer, which can be at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension. Optionally, the spacer can be cleaved from the anchor to reveal an array of deposited, single anchors comprising nucleic acid. These surface-attached anchors can optionally be used to bind/link other biological molecules or non-biological compounds to the surface to create a nanoscale array for analytical procedures.
[0009] Optionally, the anchor includes a first functional group, the surface includes a second functional group, and the anchor is linked to the surface via interaction, bonding or association between the first and second functional groups. Typically, the polymeric spacer includes a - -
polymeric molecule, e.g., DNA, RNA or protein, folded to form a three-dimensional structure. In some embodiments, the spacer of the spacer-anchor complex is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in any one dimension (e.g., length, width, height and/or diameter). In some embodiments, the spacer of the spacer-anchor complex can then be cleaved and released, leaving an anchor bound to the surface.
[0010] In some embodiments, the disclosure relates to methods for preparing an array of molecules, comprising: (a) contacting a solid or semi-solid surface with an anchor including a nucleic acid molecule having a first linking group, where the anchor is linked to a spacer that includes a nucleic acid molecule and where the surface includes a second linking group, under conditions where the first and second linking groups form a bond, thereby linking the anchor to the surface. Optionally, the contacting is in the presence of a reagent that promotes bond formation between the first and second linking groups.
[0011] In still another embodiment, the anchor molecule further comprises a cleavable linker moiety, a binding partner moiety, a reporter moiety, and/or a flexible or rigid moiety.
DETAILED DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 illustrates an exemplary method for preparing a spacer-anchor complex. [0013] FIG. 2 illustrates an exemplary method for preparing a nucleic acid array using the spacer-anchor complexes.
[0014] FIG. 3 shows microscope images of spacer-anchor complexes. FIG. 3(A) shows spacer- anchor complexes stained with SYBR Green dye. FIG. 3(B) shows spacer-anchor complexes attached to a quantum dot.
[0015] FIG. 4 shows the nucleotide sequence of coliphage lambda DNA. [0016] FIG. 5 illustrates the result of an exemplary assay performed according to Example 6.
DETAILED DESCRIPTION
[0017] In some embodiments, the disclosure relates to compositions, systems, and methods for preparing surface arrays using spacers. Optionally, one or more spacers are linked to one or more anchors to form a spacer-anchor complex. The anchor optionally includes at least one functional group capable of linking the anchor to a surface. In a typical embodiment, each spacer-anchor complex includes one or more spacers linked to a single anchor.
[0018] In some embodiments, the biomolecular array is prepared by contacting one or more spacer-anchor complexes with a surface under conditions where the anchor of the spacer-anchor complex binds to or becomes otherwise linked to the surface. Optionally, the spacer is then cleaved and released, leaving behind the surface-linked anchor. The size or shape of the spacer can optionally be adjusted to modulate the resulting spacing between adjacent surface-linked anchors. In some embodiments, this method can be used to produce an array of single anchors, each attached to a discrete location on the surface of the array. Typically, the spacing between the surface-linked anchors is sufficient to permit the resolution of individual surface-linked anchors using any suitable detection means of choice. Optionally, the anchors include functional groups that can serve as points of attachment for biomolecules (e.g., DNA or RNA) or other non- biological molecules of interest. The surface-linked anchors can optionally be used to link these molecules of interest to the surface, thus creating a nanoscale array for analytical procedures. [0019] In one exemplary embodiment, the disclosure relates to a method for forming a molecular array, comprising: contacting one or more spacer-anchor complexes including an anchor linked to a spacer, the spacer including nucleic acid folded to form a three-dimensional shape, with a solid or semi-solid surface under conditions where the anchor of the one or more spacer-anchor complexes binds to the surface, thereby linking the one or more spacer-anchor complexes to the surface. Optionally, the method further includes cleaving the linkage between the spacer and the anchor to release the spacer from the spacer-anchor complex, thus creating an array of surface- attached anchors.
[0020] In an exemplary embodiment, the spacer comprises nucleic acid folded to form a three- dimensional shape. The nucleic acid of the spacer can be double stranded or single stranded. In some embodiments, both the spacer and anchor comprise nucleic acid, and the nucleic acid of the anchor can be deposited or otherwise linked to the surface such that single anchors are linked to discrete locations on the surface.
[0021] In some embodiments, the disclosure relates to compositions, systems, and methods for use in preparing arrays of nucleic acid molecules using spacers linked to anchors. Optionally, the spacer comprises a long, double-stranded, nucleic acid molecule which is folded to form a three- dimensional shaped molecule. The anchor can comprise a short nucleic acid molecule. The arrays can be prepared by delivering the anchors to a solid surface using a spacer which restricts the - -
spacing between the anchors. The size of the spacer can be adjusted to facilitate a tunable spacing distance between the anchors.
Spacers
[0022] In some embodiments, the disclosure relates to spacers that are useful in preparing single molecule arrays for use in a variety of applications. In a typical embodiment, the spacer is linked to the molecules to be arrayed on a suitable surface and is sized so as to achieve the desired distance between adjacent molecules on the array. Typically, the spacer comprises one or more linear or branched polymers (e.g., nucleic acid or protein polymers) that are folded to form a three- dimensional shape. In one embodiment, the spacer comprises one or more linear, double- stranded nucleic acid molecules that are folded or collapsed to convert the linear form to a 3-dimensional form (Fig. 1). In some embodiments, the spacer measures at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2100 nm, 2250 nm, 2500 nm, 2750 nm, 3000 nm, 3500 nm, 3750 nm, 4000 nm, 4500 nm, 5000 nm, 7500 nm, or 10,000 nm in at least one dimension (e.g., length, width, height and/or diameter).
[0023] In some embodiments, the spacer comprises one or more nucleic acid molecules and the size of the spacer can optionally be adjusted (e.g., tuned) to be larger or smaller in any one or more dimensions (e.g., length, width, depth, and/or diameter), by varying the length of the one or more nucleic acid molecules that comprise the spacer. In some embodiments, the one or more nucleic acid molecules can be at least about 500 base pairs in length. For example, the one or more nucleic acid molecules can be at least about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 300, 350, 400, 450, 500, 600, 750, or 1000 kilobase pairs in length, or even longer. In one exemplary embodiment, the spacer comprises one or more nucleic acid molecules about 3- 200 kilobase pairs in length. The size of the spacer structure can be instrumental in controlling the spacing between adjacent spacer-anchor complexes bound to the surface of an array. Optionally, the one or more spacer-anchor complexes are bound to the surface at an average distance of between about 400 nm-2000 nm from each other, typically between about 500-1800 nm, even more typically between about 600-1600 nm.
[0024] The spacer can be linked to an anchor molecule. In one embodiment, a single spacer is linked to a single anchor molecule. [0025] There are many procedures, well known in the art, for making the desired length nucleic acid molecule for the spacer molecule. For example, the nucleic acid molecules can be prepared using recombinant DNA technology, fragmenting larger pieces into shorter pieces, ligating together smaller fragments (including fragments prepared using chemical synthesis methods), phage culture technology, or any combination thereof. Selection of the technique for producing the desired length may depend upon the desired fragment sizes and subsequent preparation steps. For example, the nucleic acid molecule may be nicked, sheared, or treated with an enzyme such as a restriction endonuclease or a nuclease (exonuclease). Alternatively, long nucleic acid molecules can be prepared by concatemerization, or ligating shorter pieces together. Any combination of fragmentation or elongation techniques may be practiced in any order to prepare the desired length of nucleic acid molecule. In one embodiment, the spacer can include nucleotide sequences that are about 48 kb in length, or about 48.5 kb in length, or about 48,502 bp in length. [0026] The spacer can be a double-stranded nucleic acid molecule, comprising nucleotide sequences from any source, including phage, bacterial plasmid, or vector sequences, or fragments thereof. The spacer can include concatemers of phage, plasmid, or vector sequences. The concatemers can be in a head-to-head or head-to-tail arrangement. The spacer can include concatemers of pBR322, or other plasmid sequences. The spacer can include nucleotide sequences which are coliphage, T4, T7, adenovirus, or other viral sequences. The spacer can include double- stranded, linearized sequences from phiX174 virus. In one embodiment, the spacer can include the nucleotide sequence from lambda phage genome. For example, the nucleotide sequence and restriction maps of lambda DNA are well known (Fig. 4) (Sanger, et al., 1982 Journal of MoI. Biol. 162:729-773). See for example the complete genomic sequence of the 1-strand of bacteriophage lambda in: GenBank NC_001416.1, GL9626243, and also GenBank J02459.1, Version Jo2459.1, GL215104, Accession Nos. J02459, M17233, M24325, V00636, and X00906. In another example, the lambda DNA can be from a commercially-available source (e.g., New England Biolabs, or DL Daniels, et al., 1983, in Appendix II: Complete Annotated Lambda Sequence, eds: RW Hendrix, et al., "Lambda-II", pp. 519-676).
[0027] Methods for forming three-dimensional structures having desired size dimensions and comprised of linear or branched polymers are also known in the art. For example, large nucleic acid molecules can be folded, condensed or collapsed into three dimensional forms, e.g., toroids, rods, globules, etc, when subjected to suitable conditions, including, for example, condensation increased ionic strength, increased or decreased temperature and/or or exposure to reagents such as - -
salts (e.g., magnesium chloride, sodium chloride, potassium acetate and the like), polycations, polyamines (e.g., hexamine cobalt (III), oligolysines, etc), spermine and/or spermidine; and polymers, e.g., PEG, etc. In some embodiments, the nucleic acid molecule that comprises the spacer can be collapsed or folded to form the 3-dimensional form (see Fig. 1) using salt, spermine, spermidine, and/or polyethylene glycol (PEG). Various publications describing methods and reaction conditions suitable for collapsing nucleic acid molecules have been described, for example, in Conwell et al., Evidence that both kinetic and thermodynamic factors govern DNA toroid dimensions: effects of magnesium (II) on DNA condensation by hexamine cobalt (III), Biochem. 43:5380-7 (2004); Conwell et al., Controlling the size of nanoscale toroidal DNA condensates with status curvature and ionic strength, Proc. Natl. Acad. Sci. USA 100(16):9296- 9301 (2003); Hud & Vilfan, Toroidal DNA Condensates: unraveling the fine structure and role of nucleation in determining size, Annu. Rev. Biophys. Biomol. Struct. 34:295-318 (2005); Ubbink & Odijk, Polymer- and salt-induced toroids of hexagonal DNA, Biophys. J. 68(1):54-61 (1995); Gosule et al., DNA Condensation with Polyamines, Part I & Part II, J. MoI. Biol. 121:311-337 (1978); Murayama & Yoshikawa, Thermodynamics of the Collapsing Phase Transition in a Single Duplex DNA Molecule, J. Phys. Chem. 103: 10517-23 (1999); Hammerman et al., Salt-Dependent DNA Superhelix Diameter Studied by Small Angle Neutron Scattering Measurements and Monte Carlo Simulations, Biophys. J. 75:3057-63 (1998). Some exemplary methods of generating folded DNA structures as disclosed in Examples 1-6. Using similar methods, it has been ascertained that a double stranded DNA molecule that is about 150 kilobase pairs in length will form a folded three dimensional structure that is about 1 micron (1000 nm) in diameter (data not shown). [0028] The spacer can include wild- type or mutant sequences. The mutant sequences can include nucleotide insertions, deletions, substitutions, or rearrangements. [0029] In one embodiment, the spacer can include wild-type or mutant coliphage genomic nucleic acid molecules. In another embodiment, the spacer can include coliphage genomic nucleic acid molecules, which are isolated from wild-type or mutant host cells. For example, the spacer can include coliphage DNA, e.g., T4 genomic DNA, T7 genomic DNA, lambda genomic DNA, which includes the -indl mutation (C substituted with a T at position 37,589), the -Sam7 mutation (G substituted with an A at position 45,352), -cI857 mutation (C substituted with a T at position 37,742), or the -cI858s7 mutation (G substituted with an A at position 43,082). [0030] The spacer can included non-methylated and methylated nucleotides, or other forms of modified nucleotides. For example, the spacer molecule can be isolated from a host strain carrying one or more methylase mutations, such as dcm, dam, or EcoKI methylase. The life cycle of lambda phage, and methods for culturing and producing phage DNA is well known in the art (DL Daniels, et al, 1983 in: "Lambda II" , pgs. 469-517, and pgs. 519-674, eds. RW Hendrix, et al., Cold Spring Harbor Laboratory, Cold Spring Harbor).
[0031] The spacer can be a nucleic acid molecule having overhang ends, blunt ends, or both. The overhang ends can be generated by restriction enzyme digestion. The overhang ends can include complementary sequences (e.g., cohesive ends) or non-complementary ends. The nucleic acid molecule can be a phage DNA molecule comprising complementary, cohesive ends. In one embodiment, the spacer can include complementary cohesive ends that are about 5-20 bases in length. In another embodiment, the nucleic acid molecule comprises complementary cohesive ends that are about 12 bases in length. In yet another embodiment, the complementary cohesive ends can include lambda cohesive end sequences, such as: 5' GGGCGGCG ACCT3' and/or 5ΑGGTCGCCGCCC3'.
[0032] The nucleic acid molecules that comprise the spacers can be prepared using any method, including recombinant DNA technology such as: coliphage DNA preparation, host- amplification of vector DNA; PCR; fragment ligation; fragment scission; and/or linearizing circular molecules; or any combination thereof.
[0033] Use of a spacer comprising one or more linear or branched polymer that is folded to form a three-dimensional structure can provide several advantages. One advantage is that the size of the spacer can be adjusted by selecting the length of one or more polymers that comprise the spacer and/or adjusting the reaction conditions employed to fold, condense or otherwise collapse the polymer. Another advantage is that the nucleic acid molecule of the spacer can be bound to the anchor using reaction chemistries that will ensure that only a single anchor is bound to the spacer, thus ensuring that only a single spacer will bind to a given location on the array surface.
Anchors
[0034] In some embodiments, the disclosure relates to anchors molecules linked to spacer molecules. The anchor molecules can be linear nucleic acid molecules. The anchor molecules can be single- or double-stranded nucleic acid molecules, including DNA, RNA, DNA/RNA hybrids, or analogs thereof. Alternatively, the anchor may not include any nucleic acid but instead may include functional groups that serve as attachment sites for nucleic acids or other molecules. Once the anchors are attached to the array surface at discrete locations, the anchors can then be reacted with the nucleic acids or other molecules to form an array of discretely spaced nucleic acids or other molecules.
[0035] The spacer attached or otherwise linked to the anchor is a "spacer-anchor" complex. In the spacer-anchor complex, the spacer can be used to deliver the anchor to a solid surface to prepare an array of anchor molecules. The size/dimension of the spacers restricts the distance between the anchor molecules which are immobilized to the solid surface (see the scheme in Fig. X). The anchor can be prepared using any procedure, including: recombinant DNA technology and/or chemical synthesis.
[0036] The length of the anchor can be selected to optimize tethering, proximity, flexibility, rigidity, and/or orientation of the attached spacer. For example, the anchor can be about 5-50, or about 5-40, or about 5-30, or about 5-20, or about 5-10 nucleotides in length. In one embodiment, the anchor can be about 15-20 nucleotides in length.
[0037] The sequence of the anchor can include any nucleotide sequence, including homo- polymeric or hetero-polymeric sequences. For example, the anchor molecule can be homo- polymeric-A, homo-polymeric -G, homo-polymeric -C, homo-polymeric -T, homo-polymeric -U, or homo-polymeric-I. In another example, the anchor can include any restriction enzyme sequence. In another example, the anchor can have a hetero-polymeric sequence, such as: 5'- GGGCGGCGACCTGGGT-Biotin-dT-3 ' .
[0038] The number of spacer and anchor molecules linked to each other can be varied. For example, one anchor can be linked to one spacer, or multiple anchors can be linked to one spacer, or one anchor can be linked to multiple spacers. The spacer and anchor are linked to each other using conventional linking methods, which include: annealing over-hang ends; ligation; linking chemistry; bridge-primer hybridization; click chemistry; and binding partners. In one embodiment, if the spacer comprises an over-hang end, the anchor can include a terminal end having a sequence that is complementary to the over-hang end to facilitate annealing the spacer and anchor together. In another embodiment, the spacer and anchor can be catalytically linked together using a ligase. In yet another embodiment, the spacer and anchor can be linked together using any type of linker moiety or linking chemistry, including: NHS ester chemistry; click chemistry; or aldehyde/hydrazide chemistry. Other linking chemistry schemes are possible. The Adaptable Anchor
[0039] In one aspect, the anchors are adaptable since they can include at least one linker moiety, reporter moiety, binding partner moiety, extension moiety, and/or flexibility/rigidity moiety. For example, the anchor can include at least one suitable linker (e.g., functional groups) that can be cleaved. In another example, the anchor can include at least one suitable linker for operable linkage to the spacer, the solid surface, or to other compounds. In another example, the anchors can include at least one suitable linker for operable linkage to reporter moieties, cleavable linkers, extension moiety, and/or binding partners. In another example, the anchors can include moieties that replace one or more nucleosides and can alter the flexibility or rigidity of the anchor. The rigidity moieties can include polyimide or phenyl units. In another example, the anchor can include at least one extension moiety which serves as an extender. In another example, the anchors can include at least one suitable linker, binding partner, reporter moiety, extension moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement on the anchors and at any position on the anchors (e.g., 5' or 3' ends, or internal positions). In another example, if the anchor is a double-stranded nucleic acid molecule (e.g., DNA), then the anchor can include a sequence that is recognized for cleavage by a restriction endonuclease enzyme. [0040] In one embodiment, the 5' end of the anchors can include an amino, NHS ester, alkyne, or aldehyde functional group that mediate linkage to the spacer or solid surface. In another embodiment, the 3' end of the anchors can include an amino or aldehyde functional group for linkage to the spacer or solid surface.
[0041] In one embodiment, the anchor can include polyethylene glycol or polyethylene oxide units, which have a polymer coil volume in aqueous environments that permit the units to extend into the environment rather than curl/coil. In one embodiment, the anchor can include about 1-12 PEG or PEO units. The anchor comprising the PEG or PEO units can be prepared by employing phosphoramidite chemistry. In another embodiment, an amino-derivatized solid phase can be used to link the PEG or PEO units to the anchor molecule (see the method disclosed by Woo in U.S. Patent No. 5,625,052). In another embodiment, successive PEO units can be added to the anchor using a base-modified deoxyuridine phosphoramidite with a TFA-protected amine (e.g., LAN from Molecular Biosystems, as disclosed by Grossman in U.S. Patent No. 5,807,682). The anchor comprising the PEG or PEO units can also include an amine group (e.g., aminohexyl group) at the 5' or 3' end. The 5' or 3' end of the anchor can be linked to the spacer. The 5' or 3' end of the anchor can be linked to the solid surface. The anchor comprising the PEG or PEO units can be prepared starting from the 5' or the 3' end of the nucleic acid molecule using solid phase synthesis methods and/or employing nucleic acid synthesis equipment (e.g., ABI 394 DNA synthesizer). [0042] In one embodiment the anchor comprises the sequence: 5'-(ATCG)-S-S-TAT-biotin- (PEO)N -amine -3', where the "ATGC" can include any nucleotide sequence, and the "S-S" can include a thiol linker or a photocleavable linker, and "N" can be about 1-12 PEO units. In one embodiment, the anchor includes five PEO units.
Linkers, Linking Chemistry, and Binding Partner Pairs
[0043] The anchors comprise at least one suitable linker or chemical bond that attaches the anchor to the: spacers, reporter moieties, binding partners, flexibility/rigidity moieties, solid surfaces, and/or other compounds.
[0044] The suitable linkers, reporter moieties, binding partners, and flexibility/rigidity moieties do not interfere with the function or activity of the spacer or the anchor, or with each other. The suitable linkers can be selected to optimize proximity, length, distance, orientation, charge, or flexibility or rigidity.
[0045] The suitable linker can be linked to the spacer, reporter moieties, solid surfaces, and/or other compounds, via covalent bonding, non-covalent bonding, ionic bonding, hydrophobic interactions, or any combination thereof. Examples of non-covalent attachment includes: ionic, hydrogen bonding, dipole-dipole interactions, van der Waals interactions, ionic interactions, and hydrophobic interactions. In particular, examples of non-covalent attachment includes: nucleic acid hybridization, protein aptamer-target binding, electrostatic interaction, hydrophobic interaction, non-specific adsorption, and solvent evaporation.
[0046] The suitable linker can include a short or long spacer-arm, a hydrophilic spacer-arm, or an extended spacer-arm. The suitable linker can be rigid or flexible. The suitable linker can be linear, non-linear, branched, bifunctional, trifunctional, homofunctional, or heterofunctional. Many cleavable, and bifunctional (both homo- and hetero-bifunctional) spacer arms with varying lengths are available commercially. Some linkers have pendant side chains or pendant functional groups, or both. The suitable linker can be resistant to heat, salts, acids, bases, light, chemicals, or shearing forces or flow. The suitable linker can include multiple amino acid residues, such as a poly-arginine linker. [0047] The suitable linker can be a cleavable, self-cleavable, or fragmentable linker. The linker can be cleavable or fragmentable using temperature, enzymatic activity, chemical agent, and/or electromagnetic radiation. The linker attachment can be reversible.
Cleavable Linkers
[0048] The anchor can include at least one suitable cleavable linker to permit release of the spacer. The suitable cleavage linker can include a disulfide, silyl, amide, thioamide, ester, thioester, vicinal diol, phosphoramidite, or hemiacetal group. Other cleavable bonds include enzymatically-cleavable bonds, such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).
[0049] The photo-cleavable linkers include nitrobenzyl derivatives, phenacyl groups, and benzoin esters. Analogs of the 2-nitrobenzyl linker, and other photocleavable linkers including: 2- nitrobenzyloxycarbonyl; nitroveratryl; 1-pyrenylmethyl; 6-nitroveratryloxycarbonyl; dimethyldimethoxybenzyloxyc arbonyl ; 5 -bromo-7 -nitroindolinyl ; O -hydroxy- alpha-methyl- cinnamoyl; methyl-6-nitroveratryloxycarbonyl; methyl-6-nitropiperonyloxycarbonyl; 2- oxymethylene anthraquinone; dimethoxybenzyloxy carbonyl; 5-bromo-7 -nitroindolinyl; O- hydroxy- alpha- methyl cinnamoyl; and 2-oxymethylene anthriquinone (see: McGaIl, U.S. Patent No. 5,412,087; Pirrung, U.S. Patent No. 5,143,854; and Conrad, U.S. Patent No. 5,773,308). The photocleavable linkers can be illuminated with an electromagnetic source at about 320-800 nm, depending on the particular linker, to achieve cleavage. The self-cleaving linker can be a trimethyl lock or a quinone methide linker.
[0050] Many cleavable groups are known in the art. See for example, J.W. Walker, et al., 1997 Bioorg. Med. Chem. Lett. 7:1243-1248; R. S. Givens, et al., 1997 Journal of the American Chemical Society 119:8369-8370; R. S. Givens, et al., 1997 Journal of the American Chemical Society 119:2453-2463; Jung et al., 1983 Biochem. Biophys. Acta, 761: 152-162; Joshi et al., 1990 J. Biol. Chem., 265: 14518-14525; Zarling et al., 1980 J. Immunol, 124: 913-920; Bouizar et al., 1986 Eur. J. Biochem., 155: 141-147; Park et al., 1986 J. Biol. Chem., 261: 205-210; Browning et al., 1989 J. Immunol, 143: 1859-1867; and Korlach, U.S. Patent No. 7,033,764. The cleavable linker can be a commercially-available linker. - -
[0051] In one embodiment, the photocleavable linker is a phosphoramidite (e.g., Glen Research, catalog #10-4920-xx). In another embodiment, the cleavable disulfide linker is a thiol modifier (e.g., Glen Research, catalog #10-1936-xx).
Fragmentable Linkers
[0052] The fragmentable linker can be capable of fragmenting in an electronic cascade self- elimination reaction (Graham, U.S. published patent application No. 2006/0003383; and Lee, U.S. published patent application No. 2008/0050780). In some embodiments, the fragmentable linker comprises a trigger moiety. The trigger moiety comprises a substrate that can be cleaved or "activated" by a specified trigger agent. Activation of the trigger moiety initiates a spontaneous rearrangement that results in the fragmentation of the linker and release of the enjoined molecules (e.g., spacer and anchor). For example, the trigger moiety can initiate a ring closure mechanism or elimination reaction. Various elimination reactions, include 1,4-, 1,6- and 1,8 -elimination reactions.
[0053] Any means of activating the trigger moiety may be used. Selection of a particular means of activation, and hence the trigger moiety, may depend, in part, on the particular fragmentation reaction desired. In some embodiments, activation is based upon cleavage of the trigger moiety. The trigger moiety can include a cleavage site that is cleavable by a chemical reagent or enzyme. For example, the trigger moiety can include be a cleavage recognition site that is cleavable by a sulfatase (e.g., SO3 and analogs thereof), esterase, phosphatase, nuclease, glycosidase, lipase, esterase, protease, or catalytic antibody.
More Linkers
[0054] The suitable linker comprises about 1-100 plural valent atoms. In some embodiments, the linker moiety comprises about 1-40 plural valent atoms, or more, selected from the group consisting of C, N, O, S and P.
[0055] The suitable linker can include any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds or carbon- sulfur bonds. Exemplary linking members include a moiety that includes -C(O)NH-,
-C(O)O-, -NH-, -S-, -O-, and the like. The linkers can include a combination of moieties selected from amine, alkyl, alkylene, aryl, -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O)-, -S(O)n- , where n is 0, 1, 2, 3, 4, 5, or 6-membered monocyclic rings and optional pendant functional groups, for example sulfo, hydroxy and carboxy.
[0056] The suitable linker can include a pendant side chain or pendant functional group, or both. Examples of pendant moieties include hydrophilicity modifiers, for example solubilizing groups such as sulfo (-SO3H- or -SO3 - ). The trifunctional linker can be linked to multiple reporter moieties (the same or different reporter moieties) for dendritic amplification of the signal emitted by the reporter moieties (Graham, U.S. published patent application Nos. 2006/0003383 and 2007/0009980).
[0057] The suitable linker can be a rigid linker which can be used, for example, to improve a FRET signal by optimizing the orientation of the energy transfer dye. Examples of rigid linkers include benzyl linkers, proline or poly-proline linkers (S. Flemer, et al., 2008 Journal Org. Chem. 73:7593-7602), bis-azide linkers (M.P.L. Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkers synthesized by modifying the so-called "click" chemistry scheme that is described by Megiatto and Schuster (2008 Journal of the Am. Chem. Soc. 130:12872-12873). For example, click chemistry can include azide alkyne Huisgen cycloaddition or alkynyl linkage. [0058] The suitable linker can be capable of energy transfer, such as those disclosed by Ju in U.S. published patent application No. 2006/0057565.
[0059] In one embodiment, the anchor can include an NHS ester linkage, such as one provided by an NHS-carboxy-dT compound (e.g., Glen Research, catalog # 10-1535-xx). In another embodiment, the anchor can include an aldehyde linkage, such as one provided by a 5- formylindole-CE phosphoramidite (e.g., Glen Research, catalog #10-1934-xx) or a 5'-aldehyde- modifier C2 phosphoramidite compound (e.g., Glen Research, catalog #10-1933-xx). In yet another embodiment, the anchor can include an alkynyl linkage such as one provided by a 5'- hexynyl phosphoramidite compound (e.g., Glen Research, catalog # 10-1908-xx). The alkynyl linkage can be used for click chemistry.
[0060] In another embodiment, the 5' end of the anchor can include an amino group, such as one provided by a 3'-amino-modifier C7 CPG 500 compound (e.g., Glen Research, catalog #20-2957- xx). In another embodiment, the 3' end of the anchor can include an amino group, such as one provided by a 5'-amino-modifier C6 compound (e.g., Glen Research, catalog #10-1906-xx). [0061] In another embodiment, the anchors can include non-natural nucleotides having reactive group that will attach to a surface reactive group. For example, the non-natural nucleotides include peptide nucleic acids, locked nucleic acids, oligonucleotide phosphoramidates, and oligo- - -
2'-O-alkylribonucleotides. In one embodiment, the anchors (or capture probes) can be modified with one or more amino groups at their 5' or 3' ends, or internally, for attachment to modified surfaces. The amino group at the 5' end of the anchors includes: a simple amino group; a short or long tethering arm having one or more terminal amino groups; or an amino-modified thymidine or cytosine. The amino group at the 3' end of the nucleic acid molecule is typically initially protected by a fluorenylmethylcarbamoyl (Fmoc) group. To expose the amino group, the protecting group can be removed and acylated with an appropriate succinimidyl ester, such as an N-hydroxy succinimidyl ester (NHS ester). In another embodiment, the anchors can carry internal amino groups for binding to the solid surface. For example, 2' amino modified nucleic acid molecules can be produce by methoxyoxalamido (MOX) or succinyl (SUC) chemistry to produce nucleotide analogs having amino linkers attached at the 2' C of the sugar moiety. [0062] In another embodiment, the anchors can include succinylated nucleic acid molecules which can be attached to aminophenyl- or aminopropyl-modified surfaces (B. Joos et al., 1997 Anal. Biochem. 247: 96-101).
[0063] In another embodiment, the anchors can include a thiol group which is placed at the 5' or 3' end of the anchors. The thiol group can form reversible or irreversible disulfide bonds with the surface. The thiol attached to the 5' or 3' end of the anchors can be a phosphoramidate. The phosphoramidate can be attached to the 5' end using S-trityl-6-mercaptohexyl derivatives. [0064] In another embodiment, the anchors can be reacted with modifying reagents such as: carbodiimides (e.g., dicyclohexylcarbodiimide, DCC), carbonyldiimidazoles (e.g., carbonyldiimidazole, CDI2), or potassium periodate.
[0065] In another embodiment, the anchors can have protective photoprotective caps (Fodor, U.S. Patent No. 5,510,270) capped with a photoremovable protective group. DMT-protected anchors can be immobilized to the surface via a carboxyl bond to the 3' hydroxyl of the nucleoside moiety (Pease, U.S. Pat. No. 5,599,695; Pease et al., 1994 Proc. Natl. Acad. Sci. USA 91:5022- 5026).
[0066] In yet another embodiment, the anchors can be functionalized at their 5' ends with activated 1-O-mimethoxytrityl hexyl disulfide l'-[(2-cyanoethyl)-N,N-diisopropyl)] phosphoramidate (Rogers et al., 1999 Anal. Biochem. 266:23). Binding Partner Pairs
[0067] Binding partner pairs can be used to link the spacer, anchor, reporter moiety, flexibility/rigidity moiety, other compounds, and/or solid surface, to each other in any combination. For example, one member of the binding partner pair can be linked to one end of the anchor, and the other member of the binding partner can be linked to the solid surface. [0068] Examples of binding partners include: biotin or desthiobiotin or photoactivatable biotin and their binding partners avidin, streptavidin, NEUTRA VIDIN, or CAPTA VIDIN; His-tags which bind with nickel, cobalt or copper; cysteine, histidine, or histidine patch which bind Ni- NTA; maltose which binds with maltose binding protein (MBP); lectin-carbohydrate binding partners; calcium-calcium binding protein (CBP); acetylcholine and receptor-acetylcholine; protein A and binding partner anti-FLAG antibody; GST and binding partner glutathione; uracil DNA glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor) protein; antigen or epitope tags which binds to antibody or antibody fragments, particularly antigens such as digoxigenin, fluorescein, dinitrophenol or bromodeoxyuridine and their respective antibodies; mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound and protein A; receptor- receptor agonist or receptor antagonist; enzyme-enzyme cof actors; enzyme-enzyme inhibitors; and thyroxine-cortisol. Another binding partner for biotin is a biotin-binding protein from chicken (Hytonen, et al., BMC Structural Biology 7:8).
[0069] Other examples of binding partner pairs include: artificial biotin binding sequences, such as an AVI-TAG (Avidity LLC). In one embodiment, the artificial biotin binding sequence comprises a biotin ligase sequence. In another embodiment, the biotin binding sequence comprises the sequence (in single-letter amino acid symbols) GLNDIFEAQKIEWHE. The biotin can bind the lysine (K) residue within the artificial biotin binding sequence. The artificial biotin binding sequence can be used for site-specific and/or mono- biotinylation of proteins. See for example Chapmann- Smith and Cronan 1999 Trends Biochem Sci 24:359-363; M.A. Eisenberg, et al., 1982 J. Biol Chem 275:15167-15173; J.E. Cronan 1990 J Biol Chem 265:10327-10333; and PJ. Schatz 1993 Biotechnology 11:1138-1143.
Well Known Linking Chemistries
[0070] Any linking chemistry scheme can be used to generate reactive groups for linking together the spacer, anchor, cleavable linkers, binding partners, reporter moieties, flexibility/rigidity moieties, or other compounds, and/or solid surfaces, in any combination and in - -
any order. Typically, the reactive groups include: amine, aldehyde, hydroxyl, sulfate, carboxylate groups, and others.
[0071] For example, reacting activated esters, acyl azides, acyl halides, acyl nitriles, or carboxylic acids with amines or anilines to form carboxamide bonds. Reacting acrylamides, alkyl halides, alkyl sulfonates, aziridines, haloacetamides, or maleimides with thiols to form thioether bonds. Reacting acyl halides, acyl nitriles, anhydrides, or carboxylic acids with alcohols or phenols to form an ester bond. Reacting an aldehyde with an amine or aniline to form an imine bond. Reacting an aldehyde or ketone with a hydrazine to form a hydrazone bond. Reacting an aldehyde or ketone with a hydroxylamine to form an oxime bond. Reacting an alkyl halide with an amine or aniline to form an alkyl amine bond. Reacting alkyl halides, alkyl sulfonates, diazoalkanes, or epoxides with carboxylic acids to form an ester bond. Reacting an alkyl halides or alkyl sulfonates with an alcohol or phenol to form an ether bond. Reacting an anhydride with an amine or aniline to form a carboxamide or imide bond. Reacting an aryl halide with a thiol to form a thiophenol bond. Reacting an aryl halide with an amine to form an aryl amine bond. Reacting a boronate with a glycol to form a boronate ester bond. Reacting a carboxylic acid with a hydrazine to form a hydrazide bond. Reacting a carbodiimide with a carboxylic acid to form an N-acylurea or anhydride bond. Reacting an epoxide with a thiol to form a thioether bond. Reacting a haloplatinate with an amino or heterocyclic group to form a platinum complex. Reacting a halotriazine with an amine or aniline to form an aminotriazine bond. Reacting a halotriazines with an alcohol or phenol to form a triazinyl ether bond. Reacting an imido ester with an amine or aniline to form an amidine bond. Reacting an isocyanate with an amine or aniline to form a urea. Reacting an isocyanate with an alcohol or phenol to form a urethane bond. Reacting an isothiocyanate with an amine or aniline to form a thiourea bond. Reacting a phosphoramidate with an alcohol to form a phosphite ester bond. Reacting a silyl halide with an alcohol to form a silyl ether bond. Reacting a sulfonate ester with an amine or aniline to form an alkyl amine bond. Reacting a sulfonyl halide with an amine or aniline to form a sulfonamide bond.
[0072] The linking chemistry scheme can include "click" chemistry schemes (Gheorghe, et al., 2008 Organic Letters 10:4171-4174).
[0073] The suitable linking scheme can include reacting the components to be linked in a suitable solvent in which both are soluble. Water-insoluble substances can be chemically modified in an aprotic solvent such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble materials can be accomplished using reactive compounds to make them more readily soluble in organic solvents.
PEG Attachment
[0074] Polymers of ethylene oxide can be used to attach the spacer, anchor, cleavable linkers, binding partners, reporter moieties, flexibility/rigidity moieties, or other compounds, and/or solid surfaces, to each other in any combination. Examples of polymers of ethylene oxide include: polyethylene glycol (PEG), such as short to very long PEG; branched PEG; amino-PEG-acids; PEG-amines; PEG-hydrazines; PEG-guanidines; PEG-azides; biotin-PEG; PEG-thiols; and PEG- maleinimides. In some embodiments, PEG includes: PEG-1000, PEG-2000, PEG-12-OMe, PEG- 8-OH, PEG-12-COOH, and PEG- 12-NH2.
Capture Probes
[0075] The anchor can be immobilized to the solid surface via hybridization to a capture probe (e.g., nucleic acid probe) which is linked to the solid surface. For example, the surface may comprise capture nucleic acid probes that form complexes with the anchors. The 5' or 3' end of the anchor can hybridize to the capture probe.
[0076] The capture probes can include oligonucleotide clamps (U.S. Pat. No. 5,473,060). The parameters for selecting the length and sequence of the capture probes are well known (Wetmur 1991 Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259; Britten and Davidson, chapter 1 in: Nucleic Acid Hybridization: A Practical Approach, Hames et al, editors, IRL Press, Oxford, 1985). The length and sequence of the capture probes may be selected for sufficiently stability during low and/or high stringency wash steps. The length of the capture probes ranges from about 6 to 50 nucleotides, or from about 10 to 24 nucleotides, or longer. [0077] The capture probes can be immobilized to the surface via a single or multiple biotin/avidin interactions. In one embodiment, a dual anchor can be used to immobilize the capture probe and anchor to the solid surface. The 5' or 3' end of the anchor or capture probe can be linked to a biotin molecule. The solid surface can be linked to avidin-liked molecules (e.g., avidin). The avidin molecules are capable of binding up to four biotin molecules, permitting stable binding of a biotin end-labeled duplex (e.g., capture probe/anchor) (Buzby, U.S. Patent No. 7,220,549). Linking Groups on Solid Surfaces
[0078] In one embodiment, the surface can be modified to bind amino-modified anchors. For example, 5' amino-modified nucleic acid molecules can be attached to surfaces modified with silane, such as epoxy silane derivatives (J. B. Lamture, et al., 1994 Nucleic Acids Res. 22:2121- 2125; W. G. Beattie et al., 1995 MoI. Biotechnol. 4:213-225) or isothiocyanate (Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465). Acylating reagents can be used to modify the surface for attaching the amino-modified anchors. The acylating reagents include: isothiocyanates, succinimidyl ester, and sulfonyl chloride. The amino-modified anchors can attach to surface amino groups which have been converted to amino reactive phenylisothiocyanate groups by treating the surface with p-phenylene 1,4 diisothiocyanate (PDC). In other methods, the surface amino groups can be reacted with homobifunctional crosslinking agents, such as disuccinimidylcaronate (DCS), disuccinimidyloxalate (DSO), phenylenediisothiocyanate (PDITC) or dimethylsuberimidate (DMS) for attachment to the amino-modified nucleic acid molecules. In another example, metal and metal oxide surfaces can be modified with an alkoxysilane, such as 3- aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GOPMS). [0079] In another embodiment, the surface can be treated with an alkylating agent such as iodoacetamide or maleimide for linking with thiol-modified nucleic acid molecules. [0080] In another embodiment, silane-treated surfaces (e.g., glass) can be attached with thiol- modified nucleic acid molecules using succinimidyl 4-(malemidophenyl)butyrate (SMPB). [0081] In another embodiment, thiol-modified surfaces can be used to attach anchors carrying disulfide groups (Y. H. Rogers et al., 1999 Anal. Biochem. 266:23-30).
[0082] In yet another embodiment, the surface can be coated with a polyelectrolyte multilayer (PEM) via light-directed attachment (U.S. Patent Nos. 5,599,695, 5,831,070, and 5,959,837) or via chemical attachment. The PEM chemical attachment can occur by sequential addition of polycations and polyanions (Decher, et al., 1992 Thin Solid Films 210:831-835). In one embodiment, the glass surface can be coated with a polyelectrolyte multilayer which terminated with polyanions or polycations. The polyelectrolyte multilayer can be coated with biotin and an avidin-like compound. Biotinylated molecules (nucleic acid molecules or nanocrystals) can be attached to the PEM/biotin/avidin coated surface (Quake, U.S. Patent Nos. 6,818,395, 6,911,345, and 7,501,245).
[0083] In still another embodiment, the surface is coated with a compound that increases electrostatic interaction between the surface and nucleic acid molecules (e.g., anchors and capture probes). The surface can be coated with poly-D-lysine or 3-aminopropyltriethoxysilane (Schwartz, U.S. Patent Nos. 6,221,592, 6,294,136; and Schwartz, U.S. published patent application Nos. 2006/275806 and 2007/0161028).
[0084] The surface can be coated with one or more linking agents, including: symmetrical bifunctional reagents, such as bis succinimide (e.g., bis-N-hydroxy succinimide) and maleimide (bis-N-hydroxy maleimide) esters, or toluene diisocyanate. The linking agents can be heterobifunctional cross-linkers including: m-maleimido benzoyl-N-hydroxy succinimidyl ester (MBS); succinimidyl-4-(p-maleimido phenyl)-Butyrate (SMPB); and succinimidyl-4-(N- Maleimidomethyl)Cyclohexane-l-Carboxylate (SMCC) (L. A. Chrisey et al., 1996 Nucleic Acids Res. 24:3031-3039).
[0085] In one example, a glass surface can be layered with a gold (e.g., about 2 nm layer) which is reacted with mercaptohexanoic acid. The mercaptohexanoic acid can be placed in a patterned array. The mercaptohexanoic acid can be reacted with PEG. The PEG can be reacted to bind the anchors. Any of these procedures can be used to link the solid surface to the anchors (or capture probes).
Reporter Moieties
[0086] The anchor can be linked to at least one reporter moiety. The reporter moiety generates, or causes to generate, a detectable signal. The reporter moiety can be used to locate the anchor (e.g., locate the immobilized anchor on the solid surface).
[0087] Any suitable reporter moiety may be used, including luminescent, photoluminescent, electroluminescent, bioluminescent, chemiluminescent, fluorescent (including energy transfer), phosphorescent, chromophore, radioisotope, electrochemical, mass spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. The reporter moiety generates a detectable signal resulting from a chemical or physical change (e.g., heat, light, electrical, pH, salt concentration, enzymatic activity, or proximity events). A proximity event includes two reporter moieties approaching each other, or associating with each other, or binding each other.
[0088] The reporter moieties may be selected so that each absorbs excitation radiation and/or emits fluorescence at a wavelength distinguishable from the other reporter moieties to permit monitoring the presence of different reporter moieties in the same reaction. Two or more different reporter moieties can be selected having spectrally distinct emission profiles, or having minimal overlapping spectral emission profiles. [0089] In one aspect, the signals from the different reporter moieties do not significantly overlap or interfere, by quenching, colorimetric interference, or spectral interference. [0090] The chromophore moiety may be 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, or p-nitrophenyl phosphate, and derivatives thereof.
[0091] The chemiluminescent moiety may be a phosphatase-activated 1,2-dioxetane compound. The 1,2-dioxetane compound includes disodium 2-chloro-5-(4-methoxyspiro[l,2-dioxetane-3,2'- (5-chloro-)tricyclo[3,3,l-l3'7 ]-decan]-l-yl)-l -phenyl phosphate (e.g., CDP-STAR) , chloroadamant-2'-ylidenemethoxyphenoxy phosphorylated dioxetane (e.g., CSPD) , and 3-(T- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (e.g., AMPPD). [0092] The fluorescent moiety includes: rhodols; resorufins; coumarins; xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins; phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles; stilbenes; squarenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), including Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547, DY- 630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE ; TEXAS RED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes including BODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO 465, ATTO 610 61 IX, ATTO 610 (N-succinimidyl ester), ATTO 635 (NHS ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO (7- hydroxy-9H-(l,3-dichloro-9,9-dimethylacridin-2-one or any derivatives thereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED (phosphoramidate), EPOCH YAKIMA YELLOW (phosphoramidate), EPOCH GIG HARBOR GREEN (phosphoramidate); Tokyo green (M. Kamiya, et al, 2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and CF555 (Biotium). ~ -
[0093] The fluorescent moiety can be a quencher dye, including: ATTO 540Q, ATTO 580Q, and ATTO 612Q (Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY 35 (Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate) (Glen Research). The fluorescent moiety can be a 7-hydroxycoumarin-hemicyanine hybrid molecule which is a far- red emitting dye (Richard 2008 Org. Lett. 10:4175-4178).
[0094] The fluorescent moiety may be a fluorescence-emitting metal such as a lanthanide complex, including those of Europium and Terbium.
[0095] A number of examples of fluorescent moieties are found in PCT publication WO/2008/030115, and in Haugland, Molecular Probes Handbook, (Eugene, Oregon) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999).
Energy Transfer Moieties
[0096] The anchor can be linked to an energy transfer donor and/or acceptor moiety. For example, the energy transfer donor can be a nanocrystal (e.g., quantum dot) or fluorescent dye. In another example, the energy transfer acceptor moiety can be a fluorescent dye.
[0097] The energy transfer donor is capable of absorbing electromagnetic energy (e.g., light) at a first wavelength and emitting excitation energy in response. The energy acceptor is capable of absorbing excitation energy emitted by the donor and fluorescing at a second wavelength in response.
[0098] The donor and acceptor moieties can interact with each other physically or optically in a manner that produces a detectable signal when the two moieties are in proximity with each other.
A proximity event includes two different moieties (e.g., energy transfer donor and acceptor) approaching each other, or associating with each other, or binding each other.
[0099] The donor and acceptor moieties can transfer energy in various modes, including: fluorescence resonance energy transfer (FRET) (L. Siryer j 97S Ann, Rev. Bioehem. 47: S 19-
846), scintillation proximity assays ( SPA) ( Hart and Greenwald 1979 Molecular Immunology
16:265-267: U.S. Pat. No. 4,658,649), luminescence resonance energy transfer CLRET) (G. Mathis
19^5 CHn. C hem. 41 :13^1 -1397), direct quenching (T vagi et al. 1998 Nature Biotechnology
16:49-53), chemiluminescenee energy transfer (CRETj CCampbelI and Patel 19S3 Bioehem.
Journal 216:185- 194;, bioluinirsescence resonance energy transfer ( BRET) {Y. Xu, el al,, 1999 Proc. Naύ. Acad, Sci. 96: J 51-156), and excimer formation (J, R. Lakowicz 19^9 ''Principles of Fluorescence Spectroscopy"', Kluwer Academic/Plenum Press, New York). [00100] In one aspect, the energy transfer pair (donor and acceptor) can be FRET donor and acceptor moieties. FRET is a distance-dependent radiation less transmission of excitation energy from a donor moiety to an acceptor moiety. Typically, the efficiency of FRET energy transmission is dependent on the inverse sixth-power of the separation distance between the donor and acceptor, which is approximately 10- 100 Angstroms. FRET is useful for investigating changes in proximity between and/or within biological molecules. FRET efficiency may depend on donor-acceptor distance r as 1/r6. The distance where FRET efficiency is 50% is termed R0, also known as the Forster distance. R0 is unique for each donor-acceptor combination and may be about 5 to 10 nm. The efficiency of FRET energy transfer can sometimes be dependent on energy transfer from a point to a plane which varies by the fourth power of distance separation (E, Jares- Erijrnan, εt a]., 2003 Nτat BiotechnoL 21:1387 ).
Embodiments of Spacer-Anchor Complexes
[00101] In some embodiments, the disclosed embodiments relate to spacer-anchor complexes comprising spacers linked to anchors. In one embodiment, one spacer is linked to one anchor. In another embodiment, the anchor can include at least one suitable cleavable moiety, binding partner, reporter moiety, and/or flexibility/rigidity moiety, in any combination thereof and in any arrangement on the adaptors. In another embodiment, the 5' or 3' end of the anchors can include a suitable linking moiety for attachment to the solid surface. In another embodiment, the 5' or 3' end of the anchors can be linked to the solid surface.
[00102] In one embodiment, the spacer- anchor complexes can include (listed in the 5' to 3' order): a reporter moiety, a cleavable moiety, a binding partner, and a 3' amino or aldehyde group. In another embodiment, the spacer- anchor complexes can include (listed in the 5' to 3' order): a 5' functional group (e.g., amino, NHS ester, alkyne, or aldehyde), binding partner, cleavable moiety, and reporter moiety. One skilled in the art will appreciate that other types of cleavable moieties, binding partners, reporter moieties, and/or linking moieties, are possible in any combination and in any order. ~ . <J ~
Solid Surfaces
[00103] Also disclosed are surfaces (e.g., solid surfaces) that can be linked to one or more anchors using the linking methodologies described supra. The immobilized anchors may be attached to the surface at their 5' ends or 3' ends, along their length, or along their length with a 5' or 3' portion exposed.
[00104] The immobilized anchors may be attached to the surface in a manner that renders them resistant to removal or degradation, including procedures that involve washing, flowing, temperature or pH changes, and reagent changes. In another aspect, the anchors may be reversibly attached to the surface.
[00105] The surface may be a solid surface, and includes planar surfaces, as well as concave, convex, or any combination thereof. The surface may comprise texture (e.g., etched, cavitated or bumps). The surface includes a nanoscale device, a channel, a well, bead, particle, sphere, filter, gel, or the inner walls of a capillary. The surface can be optically transparent, minimally reflective, minimally absorptive, or exhibit low fluorescence. The surface may be non-porous. The surface may be made from materials such as glass, borosilicate glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic (e.g., polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon, germanium, graphite, ceramics, silicon, semiconductor, high refractive index dielectrics, crystals, gels, polymers, or films (e.g., films of gold, silver, aluminum, or diamond).
Arrays
[00106] In some embodiments, the disclosure generally relates to arrays of immobilized anchors, which are prepared by employing spacers to deliver the anchors to the solid surface. Multiple spacer-anchor complexes are used to deliver multiple anchors (nucleic acid molecules) to the surface. Each spacer-anchor complex delivers a single nucleic acid molecule to a site/location on the surface. In the array, the arrangement and space-distance between the anchors is facilitated by the size dimensions of the spacer. Thus, the distances between the rows or columns (e.g., pitch) of immobilized anchors can be adjusted by using larger or smaller spacers. For example, spacers having dimensions of about 1-5 micron diameter can be used to deliver the anchors to the solid surface.
[00107] The immobilized anchors may be arranged in a random or ordered array on a surface. The ordered array includes rectilinear and hexagonal patterns. The surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface to create a patterned array. The anchors can be linked to the regions of the patterned array that include a functional linking moiety that bind to a functional linking moiety on the anchor. [00108] In the array, multiple anchors can be immobilized onto the surface. Each of the multiple immobilized anchors can bind, capture, or react with at least one biomolecule (e.g., nucleic acid molecules, polypeptides, carbohydrates, lipids, or reagents or compounds). Accordingly, an array of biomolecules can be prepared by binding, capturing, or reacting, the biomolecules with the multiple immobilized anchors.
[00109] In one embodiment, in the array, the anchors can each include a cleavable linker, which can be cleaved to release the spacers from the anchors. In another embodiment, the released spacers can reveal an array of immobilized nucleic acid molecules. In another embodiment, the immobilized anchors can be used to bind the biomolecules or nanocrystals (e.g., quantum dots). In another embodiment, the immobilized anchors are single-stranded nucleic acid molecules that can be used to capture (i.e., via hybridization) nucleic acid molecules, such as target molecules for sequencing (e.g., single molecule sequencing). In another embodiment, the immobilized anchors are nucleic acid molecules (single- or double- stranded) that can be used to bind to other biological molecules (e.g., polypeptides or antibodies), or bind chemical compounds, or bind drugs (e.g., candidate drugs), or bind to non-biological compounds.
Microscopic Features
[00110] The anchors can be immobilized onto a surface having microscopic features that permit manipulation and/or analysis, of biological molecules at a nanoscale level. The microscopic features can be at the micro meter size level, nano meter size level, or pico meter size level, or smaller sized levels. The microscopic features can be prepared from organic and/or inorganic compounds.
[00111] The microscopic features includes: channels, slits, cavities, pores, pillars, or loops. The microscopic features can have length, width, and height dimensions. The microscopic features can be linear or branched shaped, and/or be attached to inlet and/or outlet ports. The branched microscopic features can form a T or Y junction, or other shape and geometries.
[00112] The microscopic features can be used for delivering, binding, holding, confining, sorting, separating, enriching, mixing, reacting, streaming, flowing, washing, flushing, elongating, stretching, flushing, or washing the spacers or anchors, or reagents that react with the spacers or anchors.
[00113] The surface can include one or a plurality of microscopic features, typically more than 5,
10, 50, 100, 500, 1000, 10,000, 100,000, or 1,000,000, or more.
[00114] The dimensions of the microscopic features (e.g., trench width or depth) can be about 1 micron, or about 0.1 micron, or about 0.01 micron, or about 0.001 micron, or about 0.0001 micron. The dimensions of the microscopic features can be between about 10-25 nm, or about 25-
50 nm, or about 50-100 nm, or about 100-200 nm, or about 200-500 nm, or about 500-750 nm, or about 750-1000 nm. The microscopic features can have a trench width equal to or less than about
150 nanometers. The microscopic features can have a trench depth equal to or less than about 200 nanometers.
[00115] To prepare the arrays and/or microscopic features, the surface can be uncoated or coated with an adhesive and/or resist layer which can be applied to the surface in any order. The adhesive layer can bind/link the anchors. The resist layer may not bind/link, or exhibits decreased binding/linking, to the anchors.
[00116] The microscopic features may be prepared/fabricated from any suitable organic or inorganic compound including: amine, silane, biotin, avidin (or avidin-like compounds), PEG, protein binding partners, silicon, carbon, glass, polymer (e.g., poly-dimethylsiloxane), metals, titanium, aluminum, gold, chromium, platinum, silver, nitrides (e.g., boron nitrides), chromium, gold, synthetic vesicles, silicone, or any combination thereof.
[00117] The arrays and/or microscopic features may be prepared/fabricated using any suitable method, including: lithography; photolithography; deep UV lithography; soft lithography; diffraction gradient lithography (DGL); nanoimprint lithography (NIL); interference lithography; contact nanoprinting; self-assembled copolymer pattern transfer; spin coating; electron beam lithography; focused ion beam milling; plasma-enhanced chemical vapor deposition; electron beam evaporation; sputter deposition; bulk or surface micromachining; replication techniques such as embossing, printing, casting and injection molding; etching including nuclear track, chemical, or physical etching, reactive ion-etching, wet-etching; sacrificial layer etching; wafer bonding; channel sealing; and combinations thereof. The selection of the method for preparing the arrays or microscopic features may depend upon the desired size of the microscopic feature. For example, photolithography or deep UV lithography are typically selected to prepare microscopic features that are greater than about one micron. In another example, electron beam lithography is typically selected to prepare microscopic feature that are smaller than about one micron. [00118] For example, microscopic features can be prepared on a surface by: applying a photoresist compound to a glass surface; passivating the glass surface with a metal (e.g., aluminum or titanium) using a metal evaporation procedure; and removing the photo-resist to produce a metal- passivated glass surface having islands of glass that can be functionalized for binding the spacer- anchor complexes. The glass islands can be functionalized with PEG, amines, biotin, and/or avidin-like compounds, to bind the spacer- anchor complexes. The methods for applying and removing resists, metal-passivation on a glass surface, and chemical functionalization, are well known in the art.
[00119] The solid surface can be coupled to a light source, detector (e.g., photon detector), camera, and/or various plumbing components such as microvalves, micropumps, connecting channels, and microreservoirs for controlled flow (in and/or out) of reagents. [00120] In one embodiment, the nanoscale device includes: a flow cell; reservoirs for holding reagents; inlet ports in fluid communication with the reservoirs and flow cell for delivering the various reagents; outlet ports in fluid communication with the flow cell; photon detectors; and cameras for determining the location of a signal. The surface of the flow cell can be coated with PEM/biotin/avidin (U.S. Patent Nos. Quake, U.S. Patent Nos. 6,818,395, 6,911,345, and 7,501,245). The reagents can be pulled through the inlet or outlet ports via capillary action, or by vacuum (Lawson, U.S. published patent application No. 2008/0219890; and Harris, et al., 2008 Science 320:106-109, and Supplemental Materials and Methods from the supporting online material), or moved via a pressure-driven fluidics system. In another embodiment, the reagents can be pulled through the inlet or outlet ports using a passive vacuum source (Ulmer, U.S. patent No. 7,276,720).
[00121] In yet another embodiment, the flow cell can be a two-sided multi-channel flow cell comprising multiple independently-addressable sample channels and removable loading blocks for sample loading (Lawson, U.S. published patent application No. 2008/0219888). [00122] In still another embodiment, the solid surface can be enclosed by being surmounted with a sealing material using suitable methods. See, for example, U.S. Publication No. 2004/0197843. The surface can include a sample reservoir capable of releasing a fluid, and a waste reservoir capable of receiving a fluid, wherein both reservoirs are in fluid communication with a common reaction area. The surface can include a microfluidic area located adjacent to the nanofluidic area, and a gradient interface between the microfluidic and nanofluidic area that reduces the local entropic barrier for entry into a microscopic feature area (e.g., channels). See, for example, U.S. Patent No. 7,217,562. See also Cao, U.S. Patent No. 7,217,562 and U.S. published patent application No. 2007/0020772; and Han, U.S. Patent No. 6,635,163.
[00123] In another embodiment, the solid surface (e.g., glass) can be patterned using photoresists, and/or photolithography or electron-beam lithography. The patterned surface can be passivated by metal evaporation procedures (e.g., aluminum). The photo-resists can be removed. The exposed glass can be functionalized with biotin or amine groups, and the non-functionalized areas can be coated with PEG.
Methods for Binding Anchors to the Surface
[00124] In some embodiments, the disclosed embodiments generally relate to methods for: binding a nucleic acid molecule to a solid support; binding multiple nucleic acid molecules to a solid support; and preparing a nucleic acid molecule array. The methods can be practiced using the spacer-anchor complexes described herein. In general, the solid surface is contacted with the spacer-anchor complex to bind the anchor to the solid surface. The solid surface can have a random or organized pattern of linking groups to bind to the anchor.
[00125] The methods can be practiced using suitable conditions that permit binding of the anchor to the solid support, including parameters such as: time, temperature, pH, buffers, reagents, salts, and concentrations of the spacer-anchor complexes.
[00126] For example, the spacer-anchor complex can be contacted with the solid support for a time that is sufficient to permit binding the anchor portion to the solid support, such as about 10 minutes to 48 hours.
[00127] In another example, the spacer-anchor complex can be contacted with the solid support at a temperature that will permit binding the anchor portion to the solid support, such as about 4 - 50
0C.
[00128] In another example, the spacer-anchor complex can be contacted with the solid support at a pH that will permit binding the anchor portion to the solid support, such as about ph 4-12. The suitable pH will be dependent upon the type of linking chemistry between the anchor molecule and solid surface.
[00129] The buffer or reagents can include a source of monovalent or divalent ions. The buffer can include chelating agents such as EDTA and EGTA, and the like. Binding a Nucleic Acid Molecule to a Solid Surface
In one aspect, methods for binding a nucleic acid molecule to a solid surface, comprises the steps of: (a) contacting a solid surface with an anchor which comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double- stranded nucleic acid molecule which is 3-170 kilobases in length and which is folded to form a three-dimensional shape, wherein the solid surface comprises a second linking group which can form a bond with the first linking group; and (b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
Binding Multiple Nucleic Acid Molecule to a Solid Surface
[00130] In another aspect, methods for binding multiple nucleic acid molecules to a solid surface comprise the steps of: (a) contacting a solid surface with multiple anchors where each anchor comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double- stranded nucleic acid molecule which is 3-170 kilobases in length and which is folded to form a three-dimensional shape, wherein the solid surface comprises at least one second linking group which can form a bond with the first linking group; and (b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
Embodiments of the Methods
[00131] In one embodiment, the solid surface comprises a second linking group which is an amino group, aldehyde group, NHS-ester group, alkyne group, or one member of a binding partner pair. In another embodiment, the solid surface comprises a second linking group which is arranged on the surface as a random or organized pattern (e.g. array). [00132] In one embodiment, the nucleic acid molecule (i.e., anchor) is a single-stranded or double-stranded nucleic acid molecule. In another embodiment, the nucleic acid molecule is about 5-50 nt or bp in length. In another embodiment, the nucleic acid molecule further comprises a cleavable linker moiety, one member of a binding partner pair, a reporter moiety, and/or flexibility/rigidity moiety. In another embodiment, the cleavable linker moiety is a photocleavable linker moiety or a chemical-cleavable linker moiety. In another embodiment, the binding partner is a biotin molecule. In another embodiment, the reporter moiety is a fluorescent dye. In another embodiment, the first linking group is an amine, aldehyde, NHS -ester, alkyne, or one member of a binding partner pair.
[00133] In one embodiment, the cleavable linker moiety can be cleaved, for example after the nucleic acid molecule (i.e., anchor) binds to the solid surface. Cleaving the linker moiety and removing the spacer can reveal the single- stranded nucleic acid molecule, which is operable linked to the solid surface. In a typical spacer-anchor complex, one or more spacers are linked to one anchor. Therefore, a single spacer-anchor complex can deposit a single anchor to the solid surface. In another embodiment, the binding partner can be contacted with the other member of the binding partner pair before, during or after the nucleic acid molecule binds to the solid surface. In another embodiment, the fluorescent dye can be excited with an electromagnetic excitation source before, during or after the nucleic acid molecule binds to the solid surface. [00134] In one embodiment, the double-stranded nucleic acid molecule (i.e., spacer) comprises a coliphage DNA sequence. In another embodiment, the double- stranded nucleic acid molecule comprises a lambda DNA sequence. In another embodiment, the double-strand nucleic acid molecule that comprises the spacer can be decreased or increased in length to adjust the size of the three-dimensional shaped spacer.
[00135] In one embodiment, the solid surface is contacted with a homogeneous or heterogeneous population of spacer- anchor complexes. In another embodiment, a population of spacer- anchor complexes is a homogeneous population which comprises one type of nucleic acid molecule (anchor) which are linked to one type of double-stranded nucleic acid molecules (spacer). In another embodiment, a population of spacer-anchor complexes is a heterogeneous population which comprises one type of nucleic acid molecule (anchor) which are linked to different types of double-stranded nucleic acid molecules (spacer). In another embodiment, a population of spacer- anchor complexes is a heterogeneous population which comprises different types of nucleic acid molecules (anchor) which are linked to one type of double- stranded nucleic acid molecules (spacer). In another embodiment, the different types of nucleic acid molecules (anchor) comprise different sequences, lengths, cleavable linker moieties, members of a binding partner pair, reporter moieties, linking groups, and/or flexibility/rigidity moieties. In another embodiment, the different types of double-stranded nucleic acid molecules (spacer) comprise different sequences and/or lengths.
[00136] In one embodiment, the solid surface can be contacted with a first population of multiple spacer-anchor complexes (homogenous or heterogeneous), and subsequently contacted with a second population of multiple spacer-anchor complexes (homogeneous or heterogeneous). The solid surface can be contacted repeatedly with homogeneous or heterogeneous populations of spacer-anchor complexes.
[00137] In one embodiment, the surface can include multiple second linking groups which are arranged into a random or organized pattern (array). In another embodiment, the surface can include additional linking groups (e.g., 3rd, 4th, 5th linking groups, or more) which differ from the second linking groups. In another embodiment, the surface can include two or more different types of linking groups which are arranged into a random or organized pattern.
[00138] In one embodiment, the solid surface is contacted with a homogeneous or heterogeneous population of multiple nucleic acid molecules so that type of linking groups on the nucleic acid molecules bind to their cognate linking group on the solid surface.
[00139] In one embodiment, the solid surface comprises a chemical group that does not bind the first linking group.
[00140] In one embodiment, the method further includes the step of: contacting the solid surface with a reagent that modifies the chemical group that does not bind the first linking group (e.g., masking, blocking, and the like).
[00141] In one embodiment, the methods comprise an additional step: washing to remove the unbound nucleic acid molecules, or cleaved spacer molecules, or unbound binding partners, or binding reagents/buffers, or reagents that modify the chemical group that does not bind the first linking group.
[00142] In one embodiment, the methods further comprise an additional step: contacting the anchors with biological molecules (e.g., nucleic acid molecules, polypeptides, or antibodies) or chemical compounds or drug candidate compounds. This step can be conducted before, during, or after the anchor-spacer molecules is linked to the solid surface and/or cleaving the spacer.
Preparing Spacers and Anchors
[00143] The spacer and anchor molecules are nucleic acid molecules, which can be from any source, including chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast or ribosomal), recombinant molecules, cloned, amplified, chemically synthesized, or any combination thereof. The nucleic acid molecules can be isolated from any source including from: organisms such as phage, prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus, viruses, cells; tissues, body fluids, or synthesized nucleic acid molecules using recombinant DNA technology or chemical synthesis methods. The nucleic acid molecules can be from any commercially-available source.
[00144] The spacer and anchor molecules are nucleic acid molecules, which comprise naturally- occurring nucleotides or nucleotide analogs, or any combination thereof. Any portion of the nucleic acid molecule can include a base, sugar, and/or phosphate group analog. [00145] The spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include a sugar analog, such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev. 100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic & Medicinal Chemistry Letters vol. 7: 3013-3016), and other suitable sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36: 2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser 1999 Science 284:2118-2124.; and U.S. Pat. No. 5,558,991). The sugar moiety can be: ribosyl, 2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'-dideoxyribosyl, 2',3'- didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl, 2'-aminoribosyl, 2'-fluororibosyl, T- mercaptoriboxyl, 2'-alkylthioribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl, 3'-aminoribosyl, 3'- fluororibosyl, 3'-mercaptoriboxyl, 3'-alkylthioribosyl carbocyclic, acyclic, or other modified sugars.
[00146] The spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include a hetero cyclic base which includes substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, including naturally- occurring, substituted, modified, or engineered variants, or analogs of the same. The base is capable of forming Watson-Crick and/or Hoogstein hydrogen bonds with an appropriately complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-Δ2-isopentenyladenine (6iA), N6-Δ2-isopentenyl-2-methylthioadenine (2ms6iA), N6- methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2- thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deazaadenine (7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O4- methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosines; hydroxymethylcytosines; 5-methycytosines; base (Y); as well as methylated, glycosylated, and acylated base moieties; and the like. Additional exemplary bases can be found in Fasman, 1989, - -
Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca
Raton, Florida, and the references cited therein.
[00147] The spacer and anchor molecules are nucleic acid molecules, where the nucleotides can include phosphate group analogs, such as: phosphoramidate; phosphorothioate; phosphorodithioate; O-methylphosphoroamidite linkages; and peptide nucleic acid backbones and linkages.
[00148] Other nucleic acids analogs include those with bicyclic structures including locked nucleic acids; positive backbones; non-ionic backbones; and non-ribose backbones.
Nucleic Acid Sample Preparation
[00149] The ends and/or interior of the anchors or capture probes may be isolated and modified at their ends and/or the interior of the molecules using well known procedures, including: fragmentation, ligation, hybridization, enzymatic and/or chemical modification, conjugation with a reporter moiety, or linking to an energy transfer (donor or acceptor), or any combination of these procedures.
Sample Prep - Fragmentation
[00150] The nucleic acid molecules that comprise the spacer or anchor can be fragmented at random or specific sites using any fragmentation procedure. The nucleic acid molecules can be fragmented using mechanical force, including: shear forces (e.g., small orifice or a needle); nebulization (S. Surzycki 1990 in: "The International Conference on the Status and Future of Research on the Human Genome. Human Genome II", San Diego, CA, pp. 51; and S. J. Surzycki, 2000 in: "Basic Methods in Molecular Biology", New York, NY: Springer- Verlag); or sonication. [00151] The nucleic acid molecules can be chemically fragmented using, for example: acid- catalyzed hydrolysis of the backbone and cleavage with piperidine; internucleosomal DNA fragmentation using a copper (II) complex of 1,10-phenanthroline (o-phenanthroline, OP), CuII(OP)2 in the presence of ascorbic acid (Shui Ying Tsang 1996 Biochem. Journal 317:13-16). [00152] The nucleic acid molecules can be enzymatically fragmented using type I, II or III restriction endonucleases (N.E. Murray 2000 Microbiol. MoI. Biol. Rev. 64: 412-34; A. Pingoud and A. Jeltsch 2001 Nucleic Acids Res. 29: 3705-27; D. T. Dryden, et al, 2001 Nucleic Acids Res. 29: 3728-41; and A. Meisel, et al., 1992 Nature 355: 467-9). Enzymatic cleavage of DNA may include digestion using various ribo- and deoxyribonucleases or glycosylases. The nucleic acid molecules can be digested with DNase I or II. The nucleic acid fragments can be generated by enzymatically copying an RNA template. Fragments can be generated using processive enzymatic degradation (e.g., Sl nuclease). The enzymatic reactions can be conducted in the presence or absence of salts (e.g., Mg +, Mn +, and/or Ca +), and the pH and temperature conditions can be varied according to the desired rate of reaction and results, as is well known in the art.
Sample Prep - Modified Nucleic Acid Molecules
[00153] The 5' or 3' overhang ends of the nucleic acid molecules that comprise the spacer or anchor can be converted to blunt-ends using a "fill-in" procedure (e.g., dNTPS and DNA polymerase, Klenow, or Pfu or T4 polymerase) or using exonuclease procedure to digest away the protruding end.
[00154] The nucleic acid molecule ends can be ligated to one or more oligonucleotides using
DNA ligase or RNA ligase. The nucleic acid molecules can be hybridized to one or more oligonucleotides. The oligonucleotides can serve as linkers, anchors, bridges, clamps, anchors, or capture oligonucleotides.
[00155] The oligonucleotides can include sequences which are: enzyme recognition sequences
(e.g., restriction endonuclease recognition sites, DNA or RNA polymerase recognition sites); hybridization sites; or can include a detachable portion.
[00156] The oligonucleotide can linked to a protein-binding molecule such as biotin or streptavidin.
[00157] The nucleic acid molecules can be methylated, for example, to confer resistance to restriction enzyme digestion (e.g., EcoRI). The nucleic acid molecule ends can be phosphorylated or dephosphorylated.
[00158] A nick can be introduced into the nucleic acid molecules using, for example DNase I. A pre-designed nick site can be introduced in dsDNA using a double stranded probe, type II restriction enzyme, ligase, and dephosphorylation (Fu Dong-Jing, 1997 Nucleic Acids Research
25:677-679).
[00159] A nick can be repaired using polymerase (e.g., DNA pol I or phi29), ligase (e.g., T4 ligase) and kinase (polynucleotide kinase).
[00160] A poly tail can be added to the 3' end of the fragment using terminal transferase (e.g., polyA, polyG, polyC, polyT, or polyU). - -
[00161] The nucleic acid molecule can be modified using bisulfite treatment (e.g., disodium bisulfite) to convert unmethylated cytosines to uracils, which permits detection of methylated cytosines using, for example, methylation specific procedures (e.g., PCR or bisulfite genomic sequencing).
Sample Prep - Size Selection
[00162] The nucleic acid molecules that comprise the spacer or anchor can be size selected, or the desired nucleic acid molecules can separated from undesirable molecules, using any art-known methods, including gel electrophoresis, size exclusion chromatography (e.g., spin columns), sucrose sedimentation, or gradient centrifugation.
Sample Prep - Amplification
[00163] The nucleic acid molecules that comprise the spacer or anchor can be amplified using methods, including: polymerase chain reaction (PCR); ligation chain reaction, which is sometimes referred to as oligonucleotide ligase amplification (OLA); cycling probe technology (CPT); strand displacement assay (SDA); transcription mediated amplification (TMA); nucleic acid sequence based amplification (NASBA); rolling circle amplification (RCA); and invasive cleavage technology.
Sample Prep - Enrichment
[00164] Undesired compounds can be removed or separated from the desired nucleic acid molecules to facilitate enrichment of the desired molecules (e.g., spacers or anchors). Enrichment methods can be achieved using well known methods, including gel electrophoresis, chromatography, or solid phase immobilization (reversible or non-reversible). For example, AMPURE beads (Agencourt) can bind DNA fragments but not bind unincorporated nucleotides, free primers, DNA polymerases, and salts, thereby facilitating enrichment of the desired DNA fragments.
[00165] The desired nucleic acid molecules can be enriched using a dialysis procedure, which can be conducted by employing a dialysis membrane having a suitable molecular weight cut-off (MWCO) limit, for a sufficient amount of time, and in a suitable exchange buffer. For example, the nucleic acid molecules can be enriched using dialysis membranes having about 2K, 3.5K, 7K, or 1OK MWCO. The dialysis procedure can be conducted for about 2-48 hours. The exchange buffer can include Tris at a pH range of about pH 6-9.
EXAMPLE 1
Preparing Spacer-anchor complexes
A) Anchor Molecules:
[00166] A biotinylated oligonucleotide was used as the anchor. The oligonucleotide was diluted to 1 μM in a TE buffer (1 rnM Tris, 0.1 rnM EDTA, pH 8). The sequence of the biotinylated oligonucleotide is: [00167] pGGGCGGCGACCTGGGT-Biotin-dT.
B) Spacer Molecules:
[00168] The spacer DNA was attached to anchor DNA according to the following procedure (see scheme in Fig. 1). Lambda DNA was ligated to the biotinylated oligonucleotide in a 200 μL reaction volume: 5 μL of 1 μM biotinylated oligonucleotide; 20 μL of 1OX Taq ligase buffer (20 mM Tris-HCl, 25 mM potassium acetate, 10 mM magnesium acetate, 1 mM NAD, 10 mM dithiothreitol, 0.1 % Triton X-100; pH 7.6); 10 μL Taq ligase (New England Biolabs, #M0208L); 5 μL of 1 μg/μL lambda DNA (New England Biolabs, # N301 IS); 106 μL water. Cycle ligation was performed: (step 1) hold at 80 0C for 5 minutes; (step 2) hold at 55 0C for 8 hours; (step 3) 25 x cycles at 85 0C for 30 seconds and 55 0C for 60 seconds; (step 4) hold at 4 0C. [00169] The un-ligated oligonucleotides were removed by using a dialysis cassette (Slide-a- Lyzer, Pierce, #MWC0 10,000) in 3 exchanges of 500 mL of Tris, pH 8, for 6 hours per exchange. The dialyzed, spacer-anchor ligation product was stored at 4 0C.
C) Three-Dimensional Spacers:
[00170] The spacer DNA, while attached to anchor DNA, was collapsed to form 3-dimensional spacers (see scheme in Fig. 1) in a total 80 μL volume, at room temperature for 2 hours: 40 μL of spacer-anchor DNA from Example 1 above ; 25 μL of 50% (wt/vol) PEG 20,000 (Fluka, #95172); 5 μL of NEB buffer 3 (New England Biolabs: 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9); 8 μL of 20X SYBR Green dye; and 2 μL of streptavidin-Qdot (Invitrogen, # QlOlOlMP). - -
[00171] The 3-dimensional spacer-anchor complexes were observed under the microscope (see Figure 3). The left panel shows SYBR Green-stained spacers. The right panel shows spacer- anchor complexes labeled with Qdots in a 1:1 ratio.
EXAMPLE 2
A) Preparing Anchors with Linking Groups:
[00172] The 5' end of the anchors are functionalized for linkage to the surface by reacting with: an NHS-carboxy-dT compound (e.g., Glen Research, catalog # 10-1535-xx) for NHS-ester linkage; a 5-formylindole-CE phosphoramidite (e.g., Glen Research, catalog #10-1934-xx) or a 5'- aldehyde-modifier C2 phosphoramidite compound (e.g., Glen Research, catalog #10-1933-xx) for an aldehyde linkage; a 5'-hexynyl phosphoramidite compound (e.g., Glen Research, catalog # 10- 1908-xx) for an alkynyl linkage (e.g., for click chemistry); or a 3'-amino-modifier C7 CPG 500 compound (e.g., Glen Research, catalog #20-2957-xx) for amine linkage.
[00173] The 3' end of the anchors are functionalized for linkage to the surface by reacting with: a 5'-amino-modifier C6 compound (e.g., Glen Research, catalog #10-1906-xx) for amine linage.
B) Preparing Anchors with Cleavable Linkers:
[00174] The anchors are synthesized to include cleavable linkers using standard chemical synthesis procedures. For example, the anchors include a photocleavable linker such as 3-(4,4'- dimethoxytriyl)-l-(2-nitophenyl)-propan-l-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite (e.g., Glen Research, catalog #10-4920-xx). In another example, the anchors include a cleavable disulfide linker such as l-O-dimethoxytrityl-hexyl-disulfie,l'-{(2-cyanoethyl)- (N,N-diisopropyl)]-phosphoramidite (e.g., Glen Research, catalog #10-1936-xx).
EXAMPLE 3 Preparing Solid Surfaces
A) Homogeneous Modification:
[00175] For preparing glass slides with one functionality group or metal-patterned slides where the metal region is not interrogated. The glass slide (with or without metal-patterning) is oxygen plasma cleaned (300 watts, 5 minutes, 150 mtorr O2) before coating with aminopropyltrimethoxysilane in a YES-1224P chemical vapor deposition chamber. The amine functionalized slide can be stored in a dessicator until reacting with the NHS ester functionalized spacer-anchor complexes.
B) Heterogeneous Modification:
[00176] For preparing patterned glass slides with two orthogonal functionalities. Pattern photoresist on a glass slide using standard photolithography or electron beam lithography so that the areas designated for binding to the spacer-anchor complexes are exposed and the remainder of the slide is masked. Plasma clean the photoresist patterned slide to descum the surface and activate for surface modification (300 watts, 10 seconds, 150 mtorr O2). Functionalize the exposed glass by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate at 50 0C overnight. Remove the patterned slide, rinse with copious amounts of deionized water, and dry under vacuum. Soak the slide in a 20 mM aqueous solution of 2- aminoethylphosphonic acid overnight. Remove slide from solution and rinse with deionized water. Strip the photoresist by sonicating the slide in acetone followed by toluene. Rinse with ethanol and dry under vacuum. Functionalize the newly exposed glass surface by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate at 50 0C overnight, followed by an overnight soak in a solution of 5 mM poly(ethylene glycol) phosphonic acid in ethanol. This procedure produces amine-functionalized islands surrounds by PEG functionality on the surface.
C) Heterogeneous Modification:
[00177] For heterogeneous modification of a titanium-patterned slide (or other transition metal). Plasma clean the titanium-patterned slide (300 watts, 5 minutes, 150 mtorr O2). Soak the patterned slide overnight in a solution of 5 mM poly(ethylene glycol) phosphonic acid in ethanol. Remove the patterned slide, rinse with copious amounts of ethanol and deionized water, and dry under vacuum. Functionalize the exposed glass surface by soaking the patterned slide in an aqueous solution of 5 mM zirconium acetylacetonate at 50 0C overnight. Remove the patterned slide, rinse with copious amounts of deionized water, and dry under vacuum. Soak the slide in a 20 mM aqueous solution of 2-aminoethylphosphonic acid overnight. Remove slide from solution and rinse with deionized water and dry under vacuum. This procedure produces amine- functionalized glass islands surrounded by PEG passivated titanium. - -
D) Preparing a Solid Surface With Amine Linking Groups:
[00178] Using slides prepared by the procedure described in section C above, soak the slides in a solution of 0.1 M glutaric anhydride, 10 rnM ( para-N,N'-dimethylpyridin) DMAP and 20 rnM diisopropylethylamine (DIEA) in anhydrous toluene, overnight at room temperature. Rinse the slides with toluene and iso-propanol thoroughly and blow dry with nitrogen gas. Soak the slides in a solution of 0.5M TSTU and 0.25M DIEA in DMSO, for 30-60 minutes at room temperature. Rinse the slides with 1 mM HCl repeatedly and blow dry with nitrogen gas. This procedure produces NHS ester linking groups on the solid surface for binding to amine linking groups of the anchors.
EXAMPLE 4
Linking Anchors to the Solid Surface:
[00179] Prepare a flow cell chamber using the slide from Example 3, section D above, and tape. Pre-treat the slide surface with 100 mM NaOAc, pH 5.5, sonicate for 60 seconds. Remove the NaOAc solution by vacuum. Load the spacer-anchor complexes, in NaOAc buffer, pH 5.5 (e.g., from Example 1 above). Incubate with agitation at room temperature for 60 minutes, or longer, to permit bond formation between the NHS ester group on the glass slide and amine on the anchor.
EXAMPLE 5 Post-Binding Steps
A) Post-Binding Washing and Blocking Steps:
[00180] After the anchor portion of the spacer-anchor complex binds the surface, the slide is washed using 100 mM NaOAc, pH 5.5, four times. Add to the chamber a blocking reagent, 0.2% mPEG-12-amine (QuantaBioDesign, PN 10288), in 100 mM NaOAc, pH 5.5. Incubate for 30 minutes at room temperature. Wash the slide four times with 1 M Tris, pH 7.4. Incubate slide for 5 minutes.
B) Chemical Cleavage:
[00181] After the slide is washed and blocked, add 100 mM DTT, incubate for 10 minutes. Add fresh 100 mM DTT, incubate for 10 minutes. Repeat three times. - -
C) Post-Cleave Washing Steps:
[00182] After the chemical cleavage steps, wash the slide four times with 100 mM DTT. Wash the slide three times with distilled water. Wash the slide four times with PBS solution.
EXAMPLE 6
[00183] An adaptor comprising the following nucleic acid sequence was prepared:
[00184] 5'-(Phos) GGGCGGCGACCTG(PEG)(PEG)(Biotin-TEG)
[00185] where (Phos) = phosphate;
[00186] PEG = polyethylene glycol; and
[00187] Biotin-TEG = l-Dimethoxytrityloxy-3-O-(N-biotinyl-3-aminopropyl)- triethyleneglycolyl-glyceryl-2-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (from Glen
Research: Catalog Number: 10-1955-xx)
[00188] The adaptor was ligated to Lambda DNA as follows: 5 uL of 1 μM adaptor (in 1 mM
Tris and 0.1 mM EDTA, pH 8.0) was mixed with 20 μL of 1OX Taq ligase buffer (200 mM Tris-
HCl, 250 mM Potassium Acetate, 100 mM Magnesium Acetate, 10 mM NAD, 100 mM DTT, 1%
Triton X-100; pH 7.6), 10 μL of Taq ligase (New England Biolabs), 5 μL Lambda DNA (New
England Biolabs, Catalog # N3011S, concentration: 1 μg/ml), and 160 μL water, resulting a reaction mixture having a total volume of 200 μL. The reaction mixture was subjected to cycle ligation including the following steps:
[00189] Step 1: 800C for 5 minutes.
[00190] Step 2: 55°C for 8 hours
[00191] Step 3: 85°C for 30 seconds, followed by 55°C for 60 seconds (repeat for 25 cycles)
[00192] Step 4: 4°C overnight
[00193] The ligated mixture was dialyzed using a Slide- A-Lyzer (Pierce) 3 times in 500 mL of 10 mM Tris, pH 8.0 for 6 hours, and then stored at 4°C.
[00194] 25 μL of ligated, dialyzed DNA was mixed with 15 μL of 20% PEG, 10 μL of 1OX
NEB Buffer III (New England Biolabs) and 2 μL of 20X SYBR Green I and incubated at room temperature for 2 hours to collapse the DNA. The DNA was then bound to array surfaces according to the following method:
[00195] Glass slides were washed once in Wash Buffer (50 mM Tris HCl, pH 7.5, 50 mM NaCl,
0.5% BSA), and then incubated with a solution of 50 nM Streptavidin (Invitrogen, Catalog # 43-
4302) in Wash Buffer at room temperature for 20 minutes. The slides were then washed three times with Wash Buffer. The ligated DNA mixture was added to the surface of each slide and agitated for one hour at room temperature. The slide surfaces were then washed three times with wash buffer. SYBR Green I (diluted to 0.5X in Wash Buffer) was then added to the slide surfaces, and the binding of DNA to the surface was then observed using an Olympus microscope at 532 nm. Representative results of slides bound to DNA (left panel), plus control slides to which no streptavidin was added (right panel) are shown in FIG. 6.

Claims

What is claimed:
1. A method for forming a molecular array, comprising: contacting one or more spacer-anchor complexes with a solid or semi-solid surface, each spacer-anchor complex including a spacer linked to an anchor, the spacer including one or more nucleic acids folded to form a three-dimensional structure, and the contacting being performed under conditions where the one or more spacer-anchor complexes bind to the surface.
2. The method of claim 1, wherein the one or more nucleic acids of the spacer are about 50-200 kilobase pairs in length.
3. The method of claim 1, wherein the spacer is at least about 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 1000 nm in any one dimension.
4. The method of claim 1, wherein the one or more spacer- anchor complexes are bound to the surface at an average distance of at least about 450 nm from each other.
5. The method of claim 1, further including the step of contacting the surface-bound spacer-anchor complexes with one or more target nucleic acid molecules under conditions where each of the one or more complexes binds to a single target nucleic acid molecule, thereby linking the single target nucleic acid molecule to the surface.
6. The method of claim 5, further including cleaving the linkage between the spacer and the anchor to release the spacer from the spacer-anchor complex.
7. The method of claim 1, further including cleaving the linkage between the spacer and the anchor to release the spacer from the spacer-anchor complex.
8. The method of claim 7, further including the step of contacting the surface-attached anchor with one or more target nucleic acid molecules under conditions where the surface- attached anchor binds to a single target nucleic acid molecule, thereby linking the target nucleic acid molecule to the surface. - -
9. The method of claim 1, wherein the anchor comprises a single-stranded or double- stranded nucleic acid molecule.
10. The method of claim 1, wherein the anchor further comprises a functional linking group selected from the group consisting of: an amino group, aldehyde group, NHS ester group and an alkyne group, and where the functional linking group links the complex to the surface or to the target nucleic acid molecule.
11. The method of claim 1, wherein the anchor further comprises a cleavable linker moiety, a binding partner moiety, a reporter moiety, an extension moiety, or a flexibility/rigidity moiety.
12. The method of claim 11, wherein the cleavable linker moiety is a photo-cleavable linker moiety, a chemical-cleavable linker moiety, or an enzymatic-cleavable linker moiety.
13. An array of single target nucleic acid molecules prepared by the method of claim 5.
14. An array of single target nucleic acid molecules prepared by the method of claim 7.
15. An array of single target nucleic acid molecules prepared by the method of claim 5, wherein the target nucleic acid molecules of the array are bound to the surface at an average distance of at least about 450 nm from each other.
16. A method for binding a nucleic acid molecule to a solid surface, comprising: a) contacting a solid surface with an anchor which comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double-stranded nucleic acid molecule which is folded to form a three-dimensional shape, wherein the solid surface comprises a second linking group which can form a bond with the first linking group; and - -
b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
17. The method of claim 16, wherein one spacer is linked to one anchor.
18. The method of claim 16, wherein the spacer comprises a coliphage DNA sequence.
19. The method of claim 16, wherein the anchor comprises a single-stranded or double- stranded nucleic acid molecule.
20. The method of claim 16, wherein the first linking group is an amino, aldehyde, NHS ester, or alkyne group.
21. The method of claim 16, wherein the anchor further comprises a cleavable linker moiety, a binding partner moiety, a reporter moiety, an extension moiety, or a flexibility/rigidity moiety.
22. The method of claim 21, wherein the cleavable linker moiety is a photo-cleavable linker moiety, a chemical-cleavable linker moiety, or an enzymatic-cleavable linker moiety.
23. The method of claim 22, further comprising cleaving the cleavable linker so as to release the spacer from the anchor.
24. The method of claim 21, wherein the binding partner is biotin.
25. The method of claim 21, wherein the reporter moiety is a fluorescent dye.
26. The method of claim 16, wherein the second linking group is an amine or NHS ester group. - -
27. A method for binding a plurality of nucleic acid molecules to a solid surface, comprising: a) contacting a solid surface with a plurality of anchors wherein each anchor comprises a nucleic acid molecule which comprises a first linking group at one end and the other end is linked to a spacer molecule which comprises a double- stranded nucleic acid molecule which is folded to form a three-dimensional shape, wherein the solid surface comprises at least one second linking group which can form a bond with the first linking group; and b) contacting the first and second linking groups with a reagent that bonds the first and second linking groups together.
28. The method of claim 27, wherein each anchor is linked to one spacer.
29. The method of claim 27, wherein the spacer comprises a coliphage DNA sequence.
30. The method of claim 27, wherein the anchor comprises a single-stranded or double- stranded nucleic acid molecule.
31. The method of claim 27, wherein the first linking group is an amino, aldehyde, NHS ester, or alkyne group.
32. The method of claim 27, wherein the anchor further comprises a cleavable linker moiety, a binding partner moiety, a reporter moiety, an extension moiety, or a flexibility/rigidity moiety.
33. The method of claim 27, wherein the cleavable linker moiety is a photo-cleavable linker moiety, a chemical-cleavable linker moiety, or an enzymatic-cleavable linker moiety.
34. The method of claim 28, further comprising cleaving the cleavable linker so as to release the spacer from the anchor.
35. The method of claim 32, wherein the binding partner is biotin.
36. The method of claim 32, wherein the reporter moiety is a fluorescent dye.
37. The method of claim 27, wherein the second linking group is an amine or NHS ester group.
38. An array of nucleic acid molecules prepared by the method of claim 27.
39. The array of claim 38, wherein the distance between the anchors which are bound to the solid surface is restricted by the size of the three-dimensional shape of the spacer.
40. The array of claim 38, wherein the array is a random array or organized array.
41. The array of claim 38, wherein the plurality of anchors is a homogeneous or heterogeneous population of anchors.
42. The array of claim 38, wherein the plurality of spacers is a homogeneous or heterogeneous population of spacers.
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