WO2009043184A1 - Ensembles modulaires nucléotidiques et leur utilisation comme dispositifs d'administration - Google Patents

Ensembles modulaires nucléotidiques et leur utilisation comme dispositifs d'administration Download PDF

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WO2009043184A1
WO2009043184A1 PCT/CA2008/001781 CA2008001781W WO2009043184A1 WO 2009043184 A1 WO2009043184 A1 WO 2009043184A1 CA 2008001781 W CA2008001781 W CA 2008001781W WO 2009043184 A1 WO2009043184 A1 WO 2009043184A1
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oligonucleotide
organic
sequence
strands
nucleotide
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PCT/CA2008/001781
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English (en)
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Hanadi Sleiman
Faisal Aldaye
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The Royal Institution For The Advancement Of Learning/Mcgill University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • DNA polyhedra present tremendous potential for drug encapsulation and release, regulation of the folding and activity of encapsulated proteins, selective encaging of nanomaterials, and assembly of 3D-networks for catalysis and biomolecule crystallization. Very little has been described on this subject over the past fifteen years, each report presenting the synthesis of a single assembly, which illustrates the need for a systematic and straightforward method to access these materials. Moreover, none of these have been on structurally dynamic or stimuli responsive systems.
  • the invention relates to a facile method of accessing a large number of three-dimensional discrete DNA assemblies.
  • the approach involves the use of single-stranded oligonucleotide building blocks, of predefined geometry, as the faces or sides of the objects to be constructed.
  • prisms such triangular, cubic, pentameric and hexameric prisms, as well as the more complex heteroprism,biprism and nanotube assemblies are accessible.
  • the use of single-stranded DNA building blocks inherently allows for dynamic character.
  • triangular prisms capable of structural switching between three predefined lengths were generated.
  • a first aspect of the invention is directed to a nucleotide assembly comprising a ) a first ring system RS 1 of the formula
  • V 1 is an organic vertex; and xi is an integer from 1 to 8, and SS is a single strand oligonucleotide, the same or different in sequence, and selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 40 units in length; wherein each organic vertex V 1 is independently selected from the group consisting of
  • Ri A , Ri B , Ri C and Ri° is a phosphate group linked to the single strand nucleotide, SS; and one of Ri E , R] F , Rj G and Ri H is a phosphate bond linked to the single strand nucleotide; the remaining of R ⁇ R I B , R I C , R ⁇ R I E , R] F , R I ° and R 1 " are independently selected from H, hydroxyl, phosphate, halogen, and and each R 3 is independently selected from H, hydroxyl, phosphate, halogen, and wherein each single strand oligonucleotide is covalently linked to the one of Rj A , Ri B , Ri° and Ri D defined as the phosphate group of a first organic vertex V 1 and covalently linked to the one of Ri E , Ri F , R] G and Ri H defined as the phosphate group of a second organic vertex V so as to form a ring of alternating
  • V is an organic vertex; and X 2 is an integer from 1 to 8, and SS is a single strand nucleotide, the same or different in sequence, and selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 40 units in length; each organic vertex V is independently selected from the group consisting of
  • R 2 ⁇ , R 2 B , R 2 C and R 2 D is a phosphate group linked to the single strand nucleotide, SS; and one of R 2 E , R 2 F , R 2 0 and R 2 H is a phosphate bond linked to the single strand nucleotide; the remaining of R 2 A , R 2 B , R 2 C , R 2 D , R 2 E , R 2 F , R 2 G and R 2 H are independently selected from H, hydroxyl, phosphate, halogen, and Ci- 6 -alkyl; and each R 3 is independently selected from H, hydroxyl, phosphate, halogen, and Ci.
  • each single strand oligonucleotide is covalently linked to the one of R 2 A , R 2 B , R 2 C and R 2 0 defined as the phosphate group of a first organic vertex V and covalently linked to the one of R 2 , R 2 , R 2 and R 2 defined as the phosphate group of a second organic vertex V 2 so as to form a ring of alternating single strand oligonucleotides and organic vertices c) an X L number of a linking nucleotide strands, LS, each selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 100 units in length, wherein x L is an integer from 3 to 10; wherein each LS is independently selected from i) a nucleotide comprising a complementary sequence to at least a portion of nucleotide sequences SS in RS ' and a complementary sequence to at least a
  • a further aspect of the invention relates to a method of preparing a nucleotide assembly as defined supra, characterized in that said preparation is modular.
  • the method of the invention comprises preparing a 3-dimensional nucleotide assembly, characterized in that it comprises a pre-assembly of at least two modules; said method comprising i. assembly of a first ring system by covalently linking a number of at least 3 organic vertices with the same number of single stranded nucleotide chains ii. linking the first ring system with a second ring system by hybridizing at least 3 of an at least partially complementary single strand of nucleotide to each of the first and second ring systems.
  • a further aspect of the invention relates to a method of preparing a nucleotide assembly as defined supra, characterized in that said preparation is modular.
  • the method of the invention comprises preparing a DNA nanotube assembly, characterized in that it comprises a pre- assembly of at one module that will polymerize; said method comprising i. assembly of a ring system by covalently linking a number of at least 3 organic vertices with the same number of single stranded nucleotide chains ii.
  • a further aspect of the invention relates to an inclusion complex comprising i) a nucleotide assembly, as defined supra, and ii) an agent selected from the group comprising a protein and a medicinal agent.
  • a further aspect of the invention relates to a method of targeting a molecule to a tissue in need of an agent comprising an inclusion complex of the invention.
  • a further aspect of the invention relates to a method of producing DNA nanotubes comprising a multitude of geometrically well-defined triangular and square DNA modules.
  • These DNA nanotubes (nt) can exist in different geometry and stiffness. Geometry is determined by the number of vertices and their localization to the linking and rigidifying strands, stiffness is determined by the number of strands that are in double- or single stranded form.
  • Figure 1 (a) (Top panel) Single-stranded and cyclic DNA building blocks triangle 3, square 4, pentagon 5 and hexamer 6. (Bottom panel) DNA of the appropriate length, sequence, and number of vertex 1 molecules is (i) synthesized on phosphate-CPG to afford the linear analogue of triangle 3. (ii) These strands are then cyclized using a complementary template strand, and (iii) are chemically ligated to yield the final single-stranded and cyclic triangle 3, following purification from denaturing polyacrylamide gel electrophoresis, (b) Initial work was directed towards the synthesis of each respective continuous strand (i.e.
  • Figure 2 (a) Assembly of modules demonstrated by PAGE analysis of triangle 3 (lane 1), all the possible intermediates leading to construction of P3 (lanes 2-5), and of the assembly P3 (lane 6). b) P3 is unaffected by either MBN or ExoVII, P3ss was only degraded by MBN, while P3-3 was digested by both MBN and ExoVII. (+/- indicate presence/absence of enzyme) (c) FRET analysis of the formation of P3 from P3ss.
  • Figure 3 (a) Diversity: Square 4, pentagon 5 and hexagon 6 (lanes 1, 3, 5) quantitatively generated cubic P4, pentagonal P5 and hexagonal P6 prisms (lanes 2, 4, 6). (b) hexagon 6 (lane 1), assembly intermediates leading to construction of HP (lanes 2, 3), quantitative assembly of HP (lane 4). (c) hexagon 6 (lane 1), intermediates leading to BP (lanes 2-4), quantitative assembly of BP (lane 5).
  • Figure 4 (a) Switching: Strands 7, 8 and 9 are used to generate dynPIO, dynP14 and dynP20, from dynP, with respective length of 10, 14 and 20 bases. Real-time structural oscillation between these structures was achieved by regenerating the intermediate dynP using strands 10, 11 and 12. (b) The structural cycle of Fig 4a was confirmed using PAGE and FRET analysis.
  • Figure 5 (a) Template strand, and the linear analogues of 3 and 4. Denaturing PAGE analysis of the linear analogues of 3 (lane 2) and 4 (lanes 3), and of the template strand used to cyclize them (lane 1). (b) Assembly of cyclic 3 and 4. Native PAGE analysis reveals the clean templated cyclization of 3 (lane 3) and 4 (lanes 5), using their respective linear analogues (lanes 2 and 4, respectively) and the complementary template strand (lane 1).
  • Figure 6 (a) Chemical ligation of 3 and 4. Native PAGE analysis reveals the generation of a single other band of relatively retarded electrophoretic mobility when the cyclic assemblies of 3 (lane 1) and 4 (lane 3) are chemically ligated using cyanogen bromide (lanes 2 and 4, respectively). These single other bands are respectively assigned to the cyclic templates 3 and 4. (b) Enzymatic digestions of 3 and 4. The crude mixtures generated from the cyclization of 3 (lane 1) and 4 (lane 3) are enzymatically digested using ExoVII (lanes 2 and 4, respectively), which confirm the cyclic nature of the bands assigned to the single-stranded and cyclic DNA templates 3 and 4.
  • Figure 7 Construction of the triangular and square-shaped DNA nanotubes 3nt and 4nt, and of the single-stranded square DNA nanotube 4nt-ss. (a) Assembly of 3' and 4'. (b) assembly of 3nt and 4nt;
  • FIG. 8 Construction of 3' and 4'.
  • the single-stranded triangle 3 (lane 1) is sequentially titrated with the complementary strands CS1-CS3 (lanes 2-4, respectively), and with the RSl- RS3 strands to quantitatively generate a fully assembled triangular rung 3' (lane 5).
  • 4 (lane 1) is similarly titrated with CS1-CS4 (lanes 2-5, respectively), and RS1-RS4 to quantitatively generate 4' (lane 6).
  • Figure 9 AFM characterization of 3nt.
  • (a) AFM analysis of 3nt reveals the formation of extended one-dimensional DNA nantobue assemblies. Bar is 2.5 ⁇ m.
  • Figure 10 Height analysis of 3nt and 4nt.
  • Cross-sectional analysis of (a) 3nt and (b) 4nt reveals a consistent height of 2.95 nm and 3.56 nm for each respective DNA nanotube.
  • the relative height ratio between 3nt and 4nt is thus 0.83.
  • FIG. 11 Construction of Triangular-shaped DNA nanotubes, specifically 3'-hp using RS-hp.
  • the same well-defined square-shaped rung 4' is used to generated (a) fully double-stranded square DNA nanotube 4nt assemblies using four double-stranded linking strands, and (b) partially single-stranded DNA nanotube 4nt-ss assemblies using one double- stranded linking strand and three single-stranded linking strands, which cross-sectional analysis shows to be of a uniform size. Bar is 2.5 ⁇ m.
  • the oligonucleotide assemblies comprises nucleic acid units of DNA, RNA, LNA, PNA, and combinations thereof.
  • An oligonucleotide strand as defined by the present invention is defined as comprising from 1 to 100 nucleic acid units.
  • LNA is known in the art as "Locked Nucleic Acid”.
  • PNA is defined as a peptide nucleic acid.
  • the present invention relates to a concept that allows for the facile assembly of a small number of building blocks in order to quantitatively access a large number of 3D nucleic acid assemblies.
  • 3D nucleic acid assemblies include a triangular prism, a cube, pentameric and hexameric prisms, as well as more complex structures, such as a heteroprism and biprism (see Figures).
  • 3D assemblies of nucleic acids such as prisms, heteroprisms and biprisms are prepared by method of the invention.
  • the preparation method is modular in that ring systems of different sizes are assembled.
  • a ring system with 3 organic vertices is a triangular (3); a ring system with 4 organic vertices is a square (4); a ring system with 5 organic vertices is a pentagonal (5) ; a ring system with 6 organic vertices is a hexagonal (6), and so forth.
  • Two triangular ring systems when assembled together by means of three linking oligonucleotide strands, form a triangular prism (P3).
  • Two square ring systems when assembled together by means of up to four linking oligonucleotide strands, form a square prism (P4).
  • the ring system RS 1 may comprise 3 organic vertices, so as to form a triangular shaped ring. In such an embodiment, xi is 1.
  • the ring system RS 1 may comprise 4 organic vertices, so as to form a square shaped ring. In such an embodiment, X 1 is 2.
  • the ring system RS 1 may comprise 5 organic vertices, so as to form a pentagonal shaped ring. In such an embodiment, xi is 3.
  • the ring system RS 1 may comprise 6 organic vertices, so as to form a hexagonal shaped ring. In such an embodiment, xi is 4.
  • a heteroprism is an assembly wherein a ring system of one size is linked to a ring system of a second size.
  • XL is typically the lower of either (xi+2) or (x 2 +2). That is to say that, for instance, in a nucleotide assembly wherein RS 1 is triangular and RS 2 is hexagonal, X L is typically 3, i.e. (xi+2). Alternatively, in an embodiment for instance wherein RS 1 is pentagonal and RS is hexagonal, XL is typically 5 (one linking strand for the lower number of (xi+2) or (x 2 +2)) but may alternatively be 3 or 4.
  • a biprism is one wherein one of the two ring systems of a prism or heteroprism is further linked to a third ring system.
  • a biprism comprising a prism a fraction of SS of RS 1 is hybridized with LS.
  • a biprism comprising a heteroprism typically the larger of the two ring systems is further linked to a third ring system.
  • X 2 is typically X 1 + X 3 .
  • the nucleotide assemblies of the present invention are prepared in quantitative or near quantitative yield, whereas prior methods provided a limited number of assemblies in very low yields (near 1%).
  • the quantitative yields of the 3D assemblies of the present invention allow for commercially viable products, suitable for inclusion of medicines such as organic molecules and proteins.
  • the quantitative yields of the 3D assemblies of the present invention are due at least in part to the assembly order, in that a single stranded intermediate is prepared prior to rigidifying the linking oligonucleotide by hybridization with a complementary oligonucleotide strand.
  • the 3D assemblies of the present invention are prepared at relatively low temperatures thereby rendering the preparation and the compounds more easily commercially accessible.
  • the method of preparation of the 3D assemblies of the present invention is performed at a temperature at which proteins are stable, typically at less than 40 0 C.
  • the nucleotide assemblies of the present invention are for use as inclusion cages in an inclusion complex, such as for drug delivery or protein encapsulation. Once delivered, the cage can be opened by cleavage or unwinding of the linking oligonucleotides by use of an aptamer, such as a smart aptamer.
  • a first aspect of the invention relates to a method of preparing a nucleotide assembly as defined infra, characterized in that said preparation is modular.
  • the invention relates to a method of preparing a 3-dimensional nucleotide assembly, characterized in that it comprises a pre-assembly of at least two modules; said method comprising i. assembly of a first ring system by covalently linking a number of at least 3 organic vertices with the same number of single stranded nucleotide chains; and ii. linking the first ring system with a second ring system by hybridizing at least 3 of an at least partially complementary single strand of nucleotide to each of the first and second ring systems.
  • the concept relates to a modular assembly where one constructs a toolbox of cyclic single- stranded oligonucleotide building blocks triangle 3, square 4, pentagon 5 and hexagon 6 (see Fig 1)
  • the single-stranded and cyclic nature of 3, 4, 5 and 6 was confirmed using ExoVTI enzymatic digestion assays.
  • nucleotide assembly comprising a ) a first ring system RS 1 of the formula
  • V 1 is an organic vertex
  • X ⁇ is an integer from 1 to 8
  • SS is a single strand oligonucleotide, the same or different in sequence, and selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 40 units in length; wherein each organic vertex V is independently selected from the group consisting of
  • Ri A , Rj B , R ⁇ C and Ri° is a phosphate group linked to the single strand nucleotide, SS; and one of Ri E , Ri F , Ri G and Ri H is a phosphate bond linked to the single strand oligonucleotide; the remaining of Ri A , R ® , Ri C , Ri°, R I E , R I F , R] G and R] H are independently selected from H, hydroxyl, phosphate, halogen, and Ci- 6 -alkyl; and each R 3 is independently selected from H, hydroxyl, phosphate, halogen, and wherein each single strand oligonucleotide is covalently linked the one of Ri A , Ri B , R I C and Ri D defined as the phosphate group of a first organic vertex V 1 and covalently linked to the one of Ri E , R] F , Rj G and R) H defined as the phosphate group of a second
  • V is an organic vertex
  • X 2 is an integer from 1 to 8
  • SS is a single strand oligonucleotide, the same or different in sequence, and selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 40 units in length
  • each organic vertex V is independently selected from the group consisting of
  • R 2 ⁇ , R 2 B , R 2 C and R 2 D is a phosphate group linked to the single strand oligonucleotide, SS; and one of R 2 E , R 2 F , R 2 G and R 2 H is a phosphate bond linked to the single strand oligonucleotide; the remaining of R 2 A , R 2 B , R 2 C , R 2 0 , R 2 E , R 2 F , R 2 G and R 2 H are independently selected from H, hydroxyl, phosphate, halogen, and Ci- 6 -alkyl; and each R 3 is independently selected from H, hydroxyl, phosphate, halogen, and Ci- 6 -alkyl wherein each single strand oligonucleotide is covalently linked to the one of R 2 A , R 2 B , R 2 C and R 2 D defined as the phosphate group of a first organic vertex V 2 and covalently linked to the one of R
  • the single stranded intermediate can be prepared in quantitative yield.
  • the assembly may be rigidified by hydridizing to complementary strands so as to form strands comprising double stranded oligonucleotides between organic vertices and at least in part, between ring systems.
  • the nucleotide assembly may be rendered rigid by further comprising rigidifying strands of oligonucleotide which are complementary, at least in part, to a sequence of the LS which spans RS 1 and RS 2 , thereby forming a double stranded oligonucleotide sequence spanning RS 1 and RS 2 .
  • the nucleotide assembly of the invention typically comprises linking oligonucleotide strands, LS, which are each an oligonucleotide which comprises a sequence which is a complementary sequence to at least a portion of oligonucleotide sequences SS in RS 1 and further comprises a sequence which is a complementary sequence to at least a portion of oligonucleotide sequences SS in RS thereby hybridizing to SS in RS and SS in RS 2 and wherein at least one sequence within the LS complementary to at least a portion of at least one SS is located at a terminal end of the LS.
  • the linking oligonucleotide strands, LS which are each an oligonucleotide comprising a complementary sequence to at least a portion of an oligonucleotide sequence of SS in RS 1 and comprising a complementary sequence to at least a portion of an oligonucleotide sequence of SS in RS 2 thereby hybridizing to SS in RS 1 and SS in RS 2 and wherein at least one sequence within the LS complementary to at least a portion of at least one SS is located at a non-terminal end of the LS.
  • the double stranded oligonucleotide sequence spanning RS 1 and RS 2 may comprise internal loops, coiling or folds, as well as other secondary structural features of oligonucleotides.
  • the rigidifying strands of nucleotide may comprise less nucleic acid units than the number of units of the LS which span between RS 1 and RS 2 . That is to say that the rigidifying strands may be shorter in length than the length of the portion of the oligonucleotide of the linking strand which spans between RS and RS .
  • Rigidity is imparted to the assembly when at least a portion of at least one of the linking strands is hybridized with a linking strand.
  • Sufficient rigidity may be imparted when only one or two or half of the linking strands are hybridized to become double stranded.
  • rigidity may be imparted when only a portion of each of the linking strands is hybridized.
  • the rigidifying strand comprises locked nucleic acids (LNA)
  • LNA locked nucleic acids
  • a portion of the linking strand comprises locked nucleic acids (LNA)
  • rigidity is partly achieved in the single stranded portion of the linking strand. Accordingly, only a portion or none of the linking strand need be hybridized in order for adequate rigidity to be achieved.
  • the rigidifying strands of oligonucleotide may comprise more nucleic acid units than the number of units of the LS which span between RS 1 and RS 2 . That is to say that the rigidifying strands may be longer in length than the length of the portion of the oligonucleotide of the linking strand which spans between RS 1 and RS 2 . The longer rigidifying strand leads to an excess single strand oligonucleotide portion which may loosely hang from the assembly.
  • the ring systems may be covalently linked to each other through the organic vertices.
  • at least one of the remaining of Ri A , Ri , Ri , Ri D , Rj E , Ri F , Ri G and Ri" and at least one of the remaining of R 2 A , R 2 B , R 2 C , R 2 0 , R2 E , R 2 F , R_ G and R 2 H are phosphate and the LS is an oligonucleotide which covalently links RS and RS 2 via said phosphates on said vertices.
  • Said assembly will typically further comprise rigidifying oligonucleotide strands of the ring systems, each of said rigidifying strands complementary in sequence to the a sequence of SS in each ring system.
  • the oligonucleotide strands may comprise DNA, RNA, LNA, PNA, or combinations thereof.
  • the oligonucleotide strands of either of the ring systems, the linking strands, or of both, comprise DNA.
  • the oligonucleotide strands are DNA strands.
  • the rigidifying strands may comprise DNA, RNA, LNA, PNA, or combinations thereof.
  • the rigidifying strands comprise DNA.
  • the rigidifying strands are DNA.
  • Heteroprisms are highly interesting embodiments of the nucleotide assemblies of the invention.
  • the second ring system is further linked to d) a third ring system RS 3 of the formula
  • V 3 is an organic vertex; and X 3 is an integer from 1 to 8, and SS is supra; each organic vertex V 3 is as defined for V 2 ; and further comprise e) an X 3 number of a linking oligonucleotide strands, LS, each selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 100 units in length, wherein each LS is an oligonucleotide comprising a complementary sequence to at least a portion of oligonucleotide sequence SS in RS 3 and a complementary sequence to at least a portion of oligonucleotide sequence SS in RS 2 thereby hybridizing to SS in RS 3 and SS in RS 2 .
  • LS linking oligonucleotide strands
  • a method of preparing a modular nucleotide assembly comprising: a ) obtaining a first ring system RS of the formula
  • V 1 is an organic vertex; and xi is an integer from 1 to 8, and SS is a single strand oligonucleotide, the same or different in sequence, and selected from the group consisting of DNA, RNA, LNA, and PNA, or combinations of units therefrom, 1 to 40 units in length; wherein each organic vertex V 1 is independently selected from the group consisting of
  • Ri ⁇ , R) B , Ri C and Ri° is a phosphate group linked to the single strand nucleotide, SS; and one of Rj E , Ri F , Ri G and Ri H is a phosphate bond linked to the single strand oligonucleotide; the remaining of Ri A , Ri B , Ri C , Ri°, R t E , Ri F , Ri G and Ri" are independently selected from H, hydroxyl, phosphate, halogen, and Ci- ⁇ -alkyl; and each R 3 is independently selected from H, hydroxyl, phosphate, halogen, and Ci- 6 -alkyl; wherein each single strand oligonucleotide is covalently linked the one of Rj ⁇ , Ri B , R] C and Ri D defined as the phosphate group of a first organic vertex V 1 and covalently linked to the one of Ri E , Rj F , R] G and Ri H defined as the phosphat
  • the nucleotide assemblies of the invention are suited for encapsulation of various molecules such as medicines and proteins, and are further suitable for use as drug delivery devices.
  • the nucleotide sequences can be selected to be immunogenic or non-immunogenic, to be aptamers or resistant to nuclease activity.
  • the nucleotides assemblies can be coated, such as by modifying one or more nucleic acids or one or more organic vertices, such a s by PEGylation, to protect the strands against nuclease activity. PEGylation (binding polyethylene glycol to the units by conventional methods) allows for greater cell permeation.
  • the nucleotide assemblies of the invention comprise PEG groups, covalently linking to base pairs of the nucleic acid units and/or to ribose units of the nucleic acid units and/or to the organic vertices such as to any of Ri A , Ri B , Ri C , Ri D , Ri E , Ri F , R I G , R, H ,R 2 A , R 2 B , R 2 C , R 2 D , R 2 E , R 2 F , R 2 0 and R 2 H which are free.
  • PEGylation may not be necessary, such as in embodiments where the nucleotide is made to target a cell membrane, such as by selection of a suitable DNA or RNA recognition sequence of at least a portion of an oligonucleotide strand of the assembly.
  • the inclusion complex can be made to open up to release the encapsulated component, such as a drug, so as to provide a high bolus type dose directly to the targeted cell. Opening of the inclusion complex by opening of the assembly of the invention may be achieved by the use of an aptamer and a secondary molecule, such as relatively benign medicine, which causes unwinding or cleavage of the oligonucleotide sequence of the assembly. Aptamers for these secondary molecules can be prepared by conventional procedures. ,
  • prism P3 is assembled using two units of triangle 3, three linking strands (LS) and three rigidifying strands (RS) (Fig Id).
  • Polyacrylamide gel electrophoresis (PAGE) analysis revealed the clean generation of all intermediates leading to the single stranded analogue of prism P3 (P3ss), and the subsequent quantitative synthesis of fully double-stranded P3 (Fig. 2a, lane 1).
  • the connectivity of prism P3 was confirmed using enzymatic digestion assays.
  • Mung bean nuclease is selective for single-stranded DNA, while ExoVII only digests open, uncyclized, single- stranded DNA.
  • prism P3 was unaffected by either MBN or ExoVII, P3ss was digested by only MBN, while P3-3 (structure in Fig. 2c, Iane4) was fully degraded by both enzymes (Fig. 2b, lanes 8 and 9).
  • FRET fluorescence resonance energy transfer
  • Heteroprism HP contains triangle and hexagon faces, and its synthesis was thus conducted using triangle 3, hexamer 6, and three linking strands (Fig. 3b).
  • biprism BP contains triangular apices and a hexameric core, and as such was constructed from two units of 3, one unit of 6, and six linking strands (Fig 3b).
  • the clean self-assembly of HP and BP was studied sequentially using PAGE (Fig. 3b ) and confirmed enzymatically.
  • the two-step construction of biprism BP i.e.
  • DNA building blocks that are cyclic can maintain their structural integrity in their single- stranded form, and thus, their use provides for an opportunity to generate structurally dynamic addressable assemblies.
  • a triangular prism dynP that contains single stranded regions separating both triangular faces (Fig.4). This synthetic intermediate was then used to generate three well-defined triangular prisms of different lengths dynPIO, dynP14 and dynP20. Assembly of each prism was achieved using strands 7-9, capable of introducing internal loops within dynP of different lengths, while realtime oscillation between each structure is conducted using eraser strands 10-12 (Fig 4a).
  • dynPIO is converted into prism dynP14 (6.9 nm) following addition of 10 to regenerate dynP, and then 8, which incorporates 6 base internal loops within assembled dynP14.
  • This assembly is similarly converted into triangular prism dyn20 (8.9 nm) using 9 and 1 1.
  • Strands 12 regenerate the initial dynP, and complete this structural cycle (Fig. 4).
  • This is the first example of a dynamic 3D discrete DNA object that is controllably oscillated between three pre-determined dimensions.
  • DNA nanotubes (nt) are currently synthesized by vertically aligning DNA duplexes into a curved motif, followed by its closure (Rothemund et al., 2004; Mitchell et al., 2004; Park et al., 2005), or by rolling and cyclizing a two-dimensional DNA origami array (Douglas et al., 2007)
  • the method described here involves the initial construction of geometrically well- defined single stranded and cyclic DNA building blocks, such as DNA triangle 3 and square 4, with rigid organic vertices 1 (Fig 1, 5 and 6).
  • These units are then assembled longitudinally as “rungs” using linking strands, to produce nanotubes with pre-designed structures (Fig 7).
  • the structure of these rungs is what dictates the geometry of the nanotubes being constructed, and permits to control and modulate the shape and size of each nanotube, one rung at a time.
  • a single-stranded triangular template 3 is used as a scaffold to first generate a well-defined triangular building block 3' (Fig 7 a, Fig 8a). This is achieved by hybridizing 3 to three complementary DNA strands (CS), which contain sticky- end overhang cohesions, to form 3-3, followed by the addition of three RS to spatially orient each of these sticky-ends above and below the plane of triangle 3'. Three double-stranded LS of appropriate sequence then assemble the set of building blocks 3' into the well-defined triangular DNA nanotubes 3nt (Fig 7b).
  • this approach can be used to generate nanotubes of any arbitrary shape and size.
  • This method enabled the synthesis of square-shaped DNA nanotubes 4nt from the cyclic and single-stranded square template 4, via the well-defined square-shaped rung 4 f (Fig Ic, 7a and 8b).
  • This design feature enables a high synthetic yield for the final triangular DNA nanotubes 3nt.
  • An analysis of the resulting assemblies by atomic force microscopy (AFM) reveals that well-defined DNA nanotubes, extending over tens of microns, indeed form. These nanotubes are characterized along both their lateral and longitudinal axes.
  • DNA nanotubes that are laterally constructed one rung at a time can be easily modulated with respect to size and shape.
  • geometrically well-defined square DNA nanotubes can be readily accessed by starting with the square-shaped DNA template 4 (Fig 8 b). Assembly of 4 into 4' (as with 3' above) equips this template with cohesive DNA strands above and below its plane (Fig 7 and 8b). Addition of double-stranded linking strands dsLS generates square DNA nanotubes 4nt AFM analysis confirms the construction of highly uniform nanotubes, and their extension over tens of microns, and shows them to be of a uniform size (Fig 12 a).
  • this approach can be seamlessly adapted to construct a new class of designer DNA nanotubes with rungs of different sizes and shapes, with unique sets of structural and functional properties. Further, this approach constructs DNA nanotubes that can be completely double- stranded and rigid, or single-stranded and more flexible and permeable. If the linking strands that join the square rungs 4' are double stranded, well-defined DNA nanotubes 4nt that are fully double-stranded are obtained (as described above). The average length of each nanotube over which it behaves as a rigid rod, i.e.
  • nanostructures Numerous applications of these well-defined nanostructures are encompassed, such as in the growth of metallic or semiconductor nanowires of tunable size and geometry, encapsulation of proteins or nanoparticles in their double-stranded form, and release in their single -stranded form, and as interconnects with switchable persistence lengths.
  • Acetic acid, boric acid, cyanogen bromide (5M in acetonitrile), EDTA, formamide, A- morpholineethanesulfonic acid (MES), MgC12, StainsAll®, and tris(hydroxymethyl)- aminomethane (Tris) were used as purchased from Aldrich.
  • IOOOA base derivatized LCAA- CPG solid support with a loading density of 32 ⁇ mol/g for general DNA synthesis, 2000A phosphate-CPG with a loading density of 15 ⁇ mol/g, 5-ethylthiotetrazole, and reagents for automated DNA synthesis were used as purchased from ChemGenes.
  • Standard automated oligonucleotide solid-phase synthesis was performed on a Perspective Biosystems Expedite 8900 DNA synthesizer. UV-vis measurements were conducted on a Varian Cary 300 biospectrophotometer. Gel electrophoresis experiments were carried out on an acrylamide 20 X 20 cm vertical Hoefer 600 electrophoresis unit. Electroelution was performed using a Centrilutor® electroeluter from Millipore. Matrix assisted laser desorption time-of-flight (MALDI-TOF) spectra were obtained using a KOMPACT MALDI III mass spectrometer. Fluorescence experiments were conducted using a Photon Technology International TimeMaster spectrofiuorimeter (model C-720F). Temperature controlled hybridizations are conducted using a Flexigene Techne 60 well thermocycler. AFM images are either acquired on a Digital Instruments "Dimension 3100" or on an E-scope microscope (Santa Barbara, CA).
  • the set of single-stranded and cyclic DNA building blocks to be synthesized are triangle 3, square 4, pentamer 5 and hexamer 6 (Fig 1 a, top panel).
  • the approach involves synthesis of a single continuous DNA stand embedded with the appropriate number of rigid organic vertex 1 molecules (i.e. three for triangle 3, four for square 4, five for pentamer 5 and six for hexamer 6), its cyclization using the respective template stand T, and its subsequent chemical ligation to afford the respective cyclic DNA building block. This is exemplified in (Fig 1 a, bottom panel) for triangle 3.
  • Synthesis was conducted on phosphate-CPG with a loading density of 15 ⁇ mol/g and a pore size of 2OO ⁇ A. Incorporation of a phosphate into the 3' position of each strand facilitates its chemical ligation.
  • Standard DNA synthesis was conducted using typical oligonucleotide synthetic protocols, while couplings of vertex 1 using a trityl protected amidite derivative 2 were performed with extended coupling and deprotection times of 15 and 2 minutes.
  • triangle 3 is constructed by synthesizing thirty bases of the appropriate sequence that are embedded with three units of vertex 1, using 2, after positions 5, 15, and 25 (Fig Ia, bottom panel).
  • Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used to characterize the linear, uncyclized, analogous of triangle 3, square 4, pentamer 5 and hexamer 6 (along with the respective template strands T used to cyclize them).
  • MALDI- TOF MS was performed using a co-matrix composed of 6-aza-2-thiothymine and fucose, and the additive spermine. The matrix was prepared according known procedures.
  • the theoretically calculated molecular masses (MM), experimentally obtained MM, and the respective sequence of each building block are summarized in Table Ia.
  • Table Ib summarizes the sequences of the linear templates 3 and 4, and of the template strand used to cyclize the building blocks for the nanotube assemblies.
  • [M + K + ] 4t TGT CA-I -AGCCAGAT T T-I -GAGT AT GAGC-I -AGCCAACCAA-1 -GGT GA-Phosphate 15,707 7 / 15,707 0
  • Hybridizations were conducted by combining 2.2 X 10 ⁇ !0 moles of each building block with 2.2 X 10 "10 moles of the cyclization template strand, in 10 ⁇ L of MES buffer (250 mM MES and 20 mM MgCl 2 , pH of 7.6) at O 0 C for 10 minutes.
  • MES buffer 250 mM MES and 20 mM MgCl 2 , pH of 7.6
  • the ligation of these assemblies using cyanogen bromide was then conducted according to a known procedure.
  • studies were performed on a control system made up of three simple linear strands.
  • hybridized cyclic triangular, square, pentameric and hexameric building blocks were thus ligated following the addition of 10 ⁇ L of cyanogen bromide (in a total volume of 30 ⁇ L of MES buffer) and were left incubating at O 0 C for 15 minutes.
  • Analysis of the crude mixtures generated following ligation of hybridized cyclic 3, 4, 5 or 6 revealed the generation of a single other major product (not shown).
  • this product is retarded in electrophoretic mobility, when compared to the unligated linear analogues, and is assigned to the respective cyclic triangle 3, square 4, pentamer 5, and hexamer 6.
  • enzymatic digestions using ExoVII were conducted.
  • ExoVII is selective for the digestion of single-stranded DNA that is linear, and will not be effective on cyclic DNA even if it is single-stranded.
  • the linear analogue of triangle 3 was subjected to digestions using varying amounts of ExoVII.
  • 1.2 X 10 "10 moles of either of the linear strands, and 1.2 X 10 "10 moles of the template strand are mixed in 10 ⁇ L of TAmg buffer (40 mM Tris, 20 mM acetic acid, 12.5 mM MgCl 2 -OH 2 O; pH 7.8), and are left incubating at O 0 C for 10 minutes.
  • TAmg buffer 40 mM Tris, 20 mM acetic acid, 12.5 mM MgCl 2 -OH 2 O; pH 7.8
  • the templated cyclization of the linear analogue of 3 (lane 2), and of the linear analogue of 4 (lane 4) occurs quantitatively in both cases (lanes 3 and 5 respectively).
  • Ligations using cyanogen bromide are conducted according to a previously reported procedure by Carriero and co-workers.
  • cyanogen bromide 5 M in acetonitrile
  • MES 250 mM MES, 20 mM MgCl 2 , pH 7.6
  • YMlO Microcon size-exclusion centrifugal filter devices
  • Triangular prism P3 is generated following the addition of two units of triangle 3, 3a and 3b, to three linking strands (LS 1-3) that are subsequently made double-stranded using complementary rigidifying strands (RSl) Table 3.
  • Figure Ib summarizes the general assembly concept used in constructing this discrete DNA object. Hybridizations are typically conducted by combining all the DNA strands in the correct molar ratio (3 X 10 "10 moles of the final assembly) in 10 ⁇ L of TAEmg buffer, and by incubating the system at O 0 C for 15 minutes. The quantitative assembly of prism P3, as well as all the possible intermediates leading up to its construction, are analyzed using 10% native polyacrylamide gel electrophoresis (10 mA, 4 0 C, 17 hours) ( Figure 2a).
  • Mung bean nuclease is selective for the digestion of single-stranded DNA over that of double-stranded DNA by a factor of 30,000: 1. This enzyme possesses both exo- and endonuclease activity, and will thus digest any portion of DNA within the assembly that is single-stranded.
  • the exonuclease Exo VII is also selective for the digestion of single-stranded DNA, but will only digest open DNA (i.e.
  • a single linker strand (LSI) was dually end-labelled with the fluorophore and quencher ROX / BHQ-2, and was used to monitor the sequential self-assembly dynamics leading to the generation of prism P3.
  • the dually labelled probes were quantified and used as supplied from Sigma-Genosys.
  • pentameric P5 and hexameric prism P6 is conducted in a similar manner (3 X 10 ⁇ 10 moles of final assembly in lO ⁇ L TAEmg, 0 0 C, 15 minutes).
  • the quantitative assembly of P4, P5 and P6 is characterized using a 10% native polyacrylamide gel, and is confirmed following digestions with both MBN and ExoVII.
  • Triangular prism dynP capable of structural switching between three predefined lengths was constructed.
  • dynP is assembled from two units of triangle 3a, triangle 3b, and from three linking strands that are each 40 bases long.
  • This set of linking strands provides a 20 base single-stranded region that spans both ends of prism dynP. It is this partially single-stranded synthetic intermediate that is then used to generate the three well-defined fully double- stranded triangular prisms of different lengths dynPlO, dynP14 and dynP20.
  • the construction of the triangular rung 3' and the square rung 4' is conducted using a number of complementary CS and ⁇ gidifymg strands RS (Fig 7 a).
  • 3' for example, is constructed from one unit of the triangular template 3, three complementary strands containing sticky-end overhang cohesions CS1-CS3, and from three ⁇ gidifying strands that serve to o ⁇ ent each of these sticky-ends into one of two lateral directions RS1-RS3.
  • Assemblies are typically conducted by combining all DNA strands in the correct molar ratios (final assembly 1.2 X 10 "10 moles; 30 ⁇ L TAmg buffer), and by incubating at 95°C for 10 minutes followed by slowly cooling to 5°C over a period of 16 hours.
  • Table 6 summarizes the sequences of the strands used to construct 3' and 4' from 3 and 4, respectively. This process is monitored sequentially and is found to occur quantitatively at each step leading to, and including, the construction of the well-defined triangular and square rungs 3' and 4' (Fig 8a and 8b, respectively).
  • Assemblies are typically conducted in 30 ⁇ L of TAmg buffer, and involve addition of the double- stranded linking strands to the already assembled rungs 3' or 4' at 40°C, for 10 minutes, followed by the slow cooling to 5°C over a period of 16 hours.
  • the linking strands are mixed with their respective rungs, m the correct molar ratio, to generate an assembly with a final concentration of 4.0 X lO 6 mol L ' .
  • AFM analysis reveals the clean formation of the triangular and square-shaped DNA nanotubes in high yields (Fig 9a), while cross-sectional analysis show them all to be of the same size (Fig 9b).
  • the square-shaped DNA nanotubes are similarly assembled in high yields, and are of the uniform size. It is of interest to note that sample preparation of the DNA nanotubes for imaging using AFM resulted in nanotube assemblies that are always somewhat embedded on the mica surface. This problem has in fact been previously observed, and is reported by others. Cross-sectional analysis of 3nt and 4nt is therefore conducted on the phase images, and can only be used to confirm that all of the DNA nanotube assemblies are of the same size.
  • the relative ratio of the observed dimensions can be used to better ascertain the formation of triangular and square-shaped DNA nanotube assemblies of the expected size.
  • the theoretically calculated diameter of 3nt is 8.9 nm, while that of 4nt is 11 nm. Therefore, the relative diameter ratio of 3nt to 4nt is theoretically expected to be 0.81.
  • the experimentally obtained cross-sectional height analysis of the 3nt and 4nt using the height images is consistently found to be 2.95 nm and 3.56 nm for all nanotubes measured, which translates into a diameter ratio of 3nt to 4nt of 0.83 (Fig 10). This value is in good agreement with the theoretically calculated value of 0 81, and can be used to indirectly confirm the formation of triangular and square-shaped DNA nanotubes of the expected size.
  • Hairpins are incorporated into the triangular-shaped DNA nanotubes to aid in their characterization.
  • the well-defined triangular rung 3'-hp is constructed using ⁇ gidifying strands with protruding hairpins RS-hp, which generates an assembly with three hairpins at each of its corner units (Fig Ha).
  • the sequences of the modified RS-hp strands are summarized in Fig 11a; the hairpins region is colored in purple for clarity.
  • AFM sample preparation typically involves the deposition of 10 ⁇ L of the self-assembled mixture (concentration of 10 pM) onto freshly cleaved mica (dimensions 2 X 2 cm), followed by adequate evaporation to achieve complete dryness (typically 30 mins in a fumehood). Whenever possible, imaging is conducted within 24 hours in order to minimize time-dependant sample degradation.
  • AFM images are acquired in air, and at room temperature. "Tapping mode" (i.e. intermittent contact imaging) is performed at a scan rate of 1 Hz using etched silicon cantilevers with a resonance frequency of - 300 kHz, a spring constant of - 42 N/m, and a tip radius of ⁇ 10 nm. All images are acquired with medium tip oscillation damping (20-30%).

Abstract

L'invention concerne un procédé permettant d'obtenir un grand nombre d'ensembles d'ADN tridimensionnels discrets, qui implique l'utilisation de blocs de construction formés d'oligonucléotides monocaténaires de géométrie prédéfinie, qui constituent les faces ou les côtés des objets à construire. A l'aide d'un ensemble modulaire graduel, des prismes, tels que des prismes triangulaires, cubiques, pentamériques et hexamériques, ainsi que des ensembles plus complexes en hétéroprismes, biprismes et nanotubes, peuvent être obtenus. L'invention concerne l'obtention d'un grand nombre d'objets d'ADN tridimensionnels discrets, qui sont également dynamiques en réponse à des stimuli externes, et étend les applications des constructions d'ADN tridimensionnelles à de nombreux domaines des nanosciences.
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US9975916B2 (en) 2012-11-06 2018-05-22 President And Fellows Of Harvard College Compositions and methods relating to complex nucleic acid nanostructures
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014012071A1 (fr) * 2012-07-12 2014-01-16 Massachusetts Institute Of Technology Procédés et appareil d'assemblage
US9073962B2 (en) 2012-07-12 2015-07-07 Massachusetts Institute Of Technology Methods of serial assembly of DNA bricks into larger structures
US9975916B2 (en) 2012-11-06 2018-05-22 President And Fellows Of Harvard College Compositions and methods relating to complex nucleic acid nanostructures
CN106459132A (zh) * 2014-03-08 2017-02-22 哈佛学院院长及董事 由自组装的含顶点的固定角度核酸结构形成的核酸多面体
EP3116889A4 (fr) * 2014-03-08 2017-08-02 President and Fellows of Harvard College Polyedres d'acides nucleiques derives de structures d'acides nucleiques auto-assemblees contenant des sommets d'angles fixes
US10550145B2 (en) 2015-03-07 2020-02-04 President And Fellows Of Harvard College Single-stranded DNA nanostructures

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