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WO2007088406A1 - Use of dna or fragments or analogs thereof as a molecular glue - Google Patents

Use of dna or fragments or analogs thereof as a molecular glue Download PDF

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Publication number
WO2007088406A1
WO2007088406A1 PCT/IB2006/000275 IB2006000275W WO2007088406A1 WO 2007088406 A1 WO2007088406 A1 WO 2007088406A1 IB 2006000275 W IB2006000275 W IB 2006000275W WO 2007088406 A1 WO2007088406 A1 WO 2007088406A1
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WO
WIPO (PCT)
Prior art keywords
dna
objects
analogs
fragments
substrates
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PCT/IB2006/000275
Other languages
French (fr)
Inventor
Bruno Samori'
Giampaolo Zuccheri
Klaus MÜLLEN
Andreas Hermann
Original Assignee
Alma Mater Studiorum- Universita'di Bologna
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Application filed by Alma Mater Studiorum- Universita'di Bologna, MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical Alma Mater Studiorum- Universita'di Bologna
Priority to PCT/IB2006/000275 priority Critical patent/WO2007088406A1/en
Publication of WO2007088406A1 publication Critical patent/WO2007088406A1/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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention is directed to the use of deoxyribonucleic acid (in the following, DNA) , or its fragments (like, for example, single strand DNA (in the following, ⁇ ssDNA) or oligodeoxynucleotide/s (in the following, ODN/s) ) , or its analogs (like, for example, ribonucleic acid (in the following, RNA) , peptide nucleic acids (in the following, PNA) , locked nucleic acids (in the following, LNA), (L) -alpha- threofuranosyl oligonucleotides (in the following, TNA) , expanded nucleic acids (in the following, xDNA) and others) for gluing- together, on a molecular basis, microscopic or macroscopic objects/substrates.
  • the invention is directed to the above disclosed .use of DNA, or fragments or analogs thereof, for a number of
  • adhesives and glues are used in all types of manufacture. For example, adhesive bonding is applied in the construction of aircrafts and finds increasing utilization in the automotive industry with a rising number of aluminium car bodies that require glue for their assembly. In the microelectronics industry, .electrically conductive adhesives are playing an increasingly important role in the design and production of electronic chip package applications. Adhesives are of great importance also in medicine. This includes, for example, the use of UV-c ⁇ ring cements in dentistry and acrylic bond cements in orthopaedics. Biological adhesives are commonly used for blocking hemorrhage or for the purposes to seal air and body fluid leakage.
  • a surgical adhesive and hemostatic agent is fibrin glue derived from plasma coagulation proteins.
  • Another example of biological glue used for hemostasis is chitosan.
  • Nature uses proteins as well to carry out adhesion.
  • a particularly interesting example is represented by mussel adhesive proteins.
  • marine organisms have the ability to form moisture-resistant adhesive bonds unrivalled by human efforts.
  • most man- - ' made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate.
  • much effort is now paid to synthesize polypeptides that are able to reproduce the properties of mussel adhesives for applications in medicine and biotechnology.
  • fragments of DNA is used, in the following, to ' indicate, for example, natural or synthetic pieces of DNA of different length, single strands of DNA (ssDNA) of different length, natural or synthetic oligonucleotides, chains of oligodeoxynucleotides (ODNs) , triple-helix-forming oligonucleotides (TFOs) and analogs thereof.
  • TFOs are, usually, the same as (or very similar to) ODNs; simply, they have a particular sequence that makes them form triple-helices with double strand DNA targets of particular sequence (homopyrimidinic- homopurinic) .
  • analogs of DNA is used to indicate structures similar to DNA, ssDNA/s, or ODN/s made of synthetic or natural polymers, like, for example, RNA, PNA, LNA, TNA, xDNA and others where heterocyclic bases, similar or different the ones found in naturally occurring nucleic acids, are arranged, in a linear or branched fashion, on a backbone based -on a ' phosphodiester linkage or on different chemistries, where bases are attached to ribose sugar rings or other cyclic or linear linkers.
  • Crucial problems to be solved are still open ' in the present adhesive technology field, in particular, as far as the mechanochemistry of adhesives is involved. Among said problems, it is worthwhile to mention at
  • non-immunogenic adhesion in particular, when applications in bio-medicine and biotechnology are needed, or in any other contexts where the materials could come in contact with persons, e.g. on the surface of objects that can be directly handled by humans;
  • adhesion in aqueous conditions it is known that conventional glues most often require organic solvents and the very strict exclusion of water traces;
  • miniaturization of the gluing process so that either microscopic objects (for example, cells ' or the like, biological tissues and so on) or macroscopic objects (for example, surfaces of inorganic or organic objects) can be properly • ⁇ ' glued; furthermore, the glue can be used on micro-patterned macroscopic objects, to select on said objects areas to be glued 'and areas not to be glued, for example, on microscopic 5 objects; or to better define fracture dynamics during the taking-apart of glued macroscopic objects, if desired;
  • Figure 1 discloses the basic concept of the DNA glue approach, wherein two substrates (micro or macroscopic) are, firstly, functionalized with two different sets of complementary chains of ODNs and then brought in contact to each other.
  • Fig. 1 shows that the more the two ODNs are complementary (in this case, they are fully complementary) , the more strongly they will hybridize and more strongly the two substrates will be glued together.
  • Figure 2 di ⁇ closes: a), a DNA adhesion bridge; b) , "the mechanical opening of the DNA bridge in a . shearing topology (this is a non-equilibrium instant event, as revealed by a saw-tooth pattern in its single-molecule-force-spectroscopy spectrum, reporting the force vs. the extension while the two surfaces are pulled apart — • F SH is the force at which the bond breacks; c) & d) , strengthening the adhesion bridge by adding a TFO to form a triple helix [c) ] or psoralens to add covalent cross-iinks between two hybridized DNA strands [d) ] .
  • Figure 3 discloses: [a), c) , d) ] , DNA adhesion threads with sacrificial structures to cushion stresses on the contacts of the two surfaces and to modulate and maintain their interaction over great extension their interaction; b) , the unzipping of intra chain loops (this is an equilibrium reversible process that takes place at a constant force, F 0Z/ as revealed by force vs extension measurements on a single molecule) ; e) , strengthening the sacrificial structures and the adhesion bridge shearing off.
  • the present invention is directed to the use of DNA or fragments or analogs thereof as a molecular glue for gluing micro and macroscopic objects/substrates on a molecular basis.
  • said DNA or fragments or analogs thereof are selected from: natural or synthetic DNA or pieces thereof, of ' different length, single strands of DNA (ssDNA) , natural . or synthetic oligonucleotides, chains of different length of oligodeoxynucleotides (ODNs) , triple-helix-forming oligonucleotides (TFOs) , or analogues thereof; hybrid structures of DNA, ssDNA, or ODNS, or analogues thereof, with synthetic or natural polymers, like, for example, polystyrene ' , polyamides, polypeptides, silicones . and so on.
  • ssDNA single strands of DNA
  • ODNs oligodeoxynucleotides
  • TFOs triple-helix-forming oligonucleotides
  • hybrid structures of DNA, ssDNA, or ODNS, or analogues thereof with synthetic or natural polymers, like, for example, polystyrene
  • they are selected from ssDNA or ODNs or analogues thereof. Most preferably, they are selected from ODNs.
  • said objects/substrates are selected from: non-adhesive materials, like, graphite, teflon, silicon; - inorganic , materials,. like, copper, silver, palladium, platinum, mercury; organic materials, like, natural or synthetic polymers, like polydimethylsiloxane; biological materials, like, -proteinaceous or carbohydrate-based materials.
  • said objects are selected from: copper, silicon, graphite, polydimethylsiloxane.
  • Said objects can have any type of suitable shape and dimension, from the micro to the macro scale.
  • the DNA based glue of the present invention is very useful for gluing, among the others, both flat or rough surfaces of different type of materials .
  • one small copper plate of suitable form is strongly linked to a polymeric plastic surface to provide a component of an electric circuit.
  • the functionalization of said objects/substrates is carried out by first properly modifying DNA, or fragments or analogs thereof, with chemical residues that bind to the object to be glued, and then by letting react said modified DNA, or fragments or analogs thereof, with said object- to give the desired ⁇ functionalized substrate.
  • the hybridization phase of the above disclosed method is performed in aqueous conditions.
  • a "hybridization buffer 1" comprising 80 mM MOPS (pH 6.8-7.5) and 100- 500 mM NaCl or of a "hybridization buffer 2" comprising 80-120 mM MgCl 2 .
  • each surface of the materials to be glued together is prepared separately with known methods, accordingly to the type of material.
  • silver or copper surfaces are pre-cleaned (a variety of known methods can be employed) and then treated with thiol- derivatised ODNs or analogues thereof.
  • This treatment is preferably made with as concentrated as possible derivatised ODNs aqueous solutions.
  • Said treatment is carried out for a time comprised from minutes up to days, depending on a .number of parameters (the type of material/s, its thickness, its dimension; the type of application and the type and strength • of the final gluing desired, for example, reversible or not) . Preferred are times as long as possible.
  • ODNs ODNs
  • the mechanical resistance of the adhesion threads settled by the base-pairing code is controlled by the unzipping or, more commonly, by the shearing of the hybridized strands of the complementary DNA or ODNs chains (as disclosed in Fig. 2a) enclosed) . Shearing is an irreversible process with a force spectrum characterized by saw tooth profiles (as disclosed in Fig. 2b) enclosed) .
  • the mechanical resistance of said ' • DNA adhesion threads can be improved by further adding, for example, during the hybridization phase, one or -more substances able to strengthen said resistance by forming additional bonds between the complementary DNA or ODNs chains.
  • Said additional substances are preferably selected among: triple-helix forming oligonucleotides (TFOs), any crosslinking agents, like, psoralene, or others, useful to said scope, suitably selected among the commercially available crosslinking agents.
  • TFOs triple-helix forming oligonucleotides
  • any crosslinking agents like, psoralene, or others, useful to said scope, suitably selected among the commercially available crosslinking agents.
  • the mechanical resistance of the same segments of hybridized strands above described is increased by the addition of a triple- helix forming oligonucleotide (TFO) or of a chemical crosslinker, like, for example, psoralene (as disclosed in Fig. 2c) and d) enclosed).
  • TFO triple- helix forming oligonucleotide
  • chemical crosslinker like, for example, psoralene (as disclosed in Fig. 2c) and d) enclosed.
  • said additional substances can also be linked directly to one of the DNA or ODNs during the functionalization of one of the objects/substrates to be glued.
  • the force of shearing is dependent on the length of the paired ODNs and on the environment conditions: 50 pN can be considered a lower bound for the force necessary to shear, apart two short paired ODNs a few tens of base-pairs long.
  • Another mechanically relevant topology can be advantageously introduced in the DNA adhesion threads: that of intra-chain loops capable of "sacrificial" unzipping transition (as disclosed in Fig. 3a) enclosed) .
  • the ' sequence of a DNA chain makes it possible to tailor adhesion bridges between two surfaces with "sacrificial" superstructures that unfold and release the tension at forces lower than those requested to break the same adhesive bridges.
  • the mechanical properties of the adhesion could be very precisely tuned in this fashion.
  • the unzipping topology can be variously combined with the shearing topology (as disclosed in Fig. 3c) & 3d) enclosed) .
  • the DNA gluing method of the present invention is advantageously used for selectively gluing on a molecular basis either microscopic or macroscopic objects as previously described. Said method resulted particularly advantageous for gluing in a very specific way object, like silicon and graphite, that can be ⁇ prepared atomically flat.
  • ODNs The attachment of ODNs to a graphite surface is very- difficult, because said functionalization may lead to the destruction of the flatness of the graphite structure.
  • the functionalization with ODNs of large molecules like, for example, the grapitic derivative hexa-peri-hexabenzocoronene was able to give acceptable results.
  • the ODNs are synthesized using ' standard phosphoramidite chemistry for preparative ' preparations.
  • ODN synthesizer for example a "Gene-assembler” , from Pharmacia
  • two scales 150 ⁇ mol and 30 ⁇ mol
  • These quantities allowed gluing together large surface areas ' of macroscopic objects.
  • the following structural features (relevant to an efficient gluing) were screened on interplaying their empirical variation with their tailoring on the basis of force measurements at the bulk and single molecule level: (i) number of base pairs per oligonucleotide and degree of match/mismatch in hybridisation;
  • loading density i.e. amount and concentration of the ODN solution per area of substrate
  • the ODN has " been modified ' to get resistance to nucleases, following the strategies developed for the antisense technology.
  • nucleases can ensure another way of reversibility of the gluing process for biomedical applications.
  • the slow rate of this process can open further possibilities for that applications based on an adhesion that must be slowly released with time
  • DNA, or fragments or analogs thereof can be advantageously employed as molecular glue.
  • they are particularly useful for preparing glue compositions • for gluing micro and macroscopic object/substrates, that assure:

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Abstract

The present invention is directed to the use of DNA or fragments or analogs thereof for gluing together, on a molecular basis, microscopic or macroscopic ob­jects/substrates. In particular, the invention is directed to the use of DNA, or fragments or analogs thereof, for prepar­ing molecular glues for applications in different technological fields, like, biomedicine, biotechnol­ogy, electronic and automotive industry.

Description

"USE OF DNA OR FRAGMENTS OR ANALOGS THEREOF AS A
MOLECULAR GLXJE"
* * * * *
SUMMARY OF THE INVENTION The present invention is directed to the use of deoxyribonucleic acid (in the following, DNA) , or its fragments (like, for example, single strand DNA (in the following, ■ ssDNA) or oligodeoxynucleotide/s (in the following, ODN/s) ) , or its analogs (like, for example, ribonucleic acid (in the following, RNA) , peptide nucleic acids (in the following, PNA) , locked nucleic acids (in the following, LNA), (L) -alpha- threofuranosyl oligonucleotides (in the following, TNA) , expanded nucleic acids (in the following, xDNA) and others) for gluing- together, on a molecular basis, microscopic or macroscopic objects/substrates. In particular, the invention is directed to the above disclosed .use of DNA, or fragments or analogs thereof, for a number of applications in different technological fields, like," for example, biomedicine, biotechnology, the electronic and automotive industries.
TECHNICAL FIELD OF THE INVENTION Nowadays adhesives and glues are used in all types of manufacture. For example, adhesive bonding is applied in the construction of aircrafts and finds increasing utilization in the automotive industry with a rising number of aluminium car bodies that require glue for their assembly. In the microelectronics industry, .electrically conductive adhesives are playing an increasingly important role in the design and production of electronic chip package applications. Adhesives are of great importance also in medicine. This includes, for example, the use of UV-cύring cements in dentistry and acrylic bond cements in orthopaedics. Biological adhesives are commonly used for blocking hemorrhage or for the purposes to seal air and body fluid leakage. One important example of" a surgical adhesive and hemostatic agent is fibrin glue derived from plasma coagulation proteins. Another example of biological glue used for hemostasis is chitosan. Nature uses proteins as well to carry out adhesion. A particularly interesting example is represented by mussel adhesive proteins. Actually, marine organisms have the ability to form moisture-resistant adhesive bonds unrivalled by human efforts. In fact, most man- - 'made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. On consequence, much effort is now paid to synthesize polypeptides that are able to reproduce the properties of mussel adhesives for applications in medicine and biotechnology.
In this respect, DNA, or fragments or analogs thereof have not jet been utilized for gluing together microscopic or macroscopic objects. .Within the scope of the present invention, the term "fragments of DNA" is used, in the following, to ' indicate, for example, natural or synthetic pieces of DNA of different length, single strands of DNA (ssDNA) of different length, natural or synthetic oligonucleotides, chains of oligodeoxynucleotides (ODNs) , triple-helix-forming oligonucleotides (TFOs) and analogs thereof. TFOs are, usually, the same as (or very similar to) ODNs; simply, they have a particular sequence that makes them form triple-helices with double strand DNA targets of particular sequence (homopyrimidinic- homopurinic) . On its part, the term "analogs" of DNA is used to indicate structures similar to DNA, ssDNA/s, or ODN/s made of synthetic or natural polymers, like, for example, RNA, PNA, LNA, TNA, xDNA and others where heterocyclic bases, similar or different the ones found in naturally occurring nucleic acids, are arranged, in a linear or branched fashion, on a backbone based -on a' phosphodiester linkage or on different chemistries, where bases are attached to ribose sugar rings or other cyclic or linear linkers. Crucial problems to be solved are still open ' in the present adhesive technology field, in particular, as far as the mechanochemistry of adhesives is involved. Among said problems, it is worthwhile to mention at least the following ones:
(i) complete mechanical control along the whole extension of separation of the glued surfaces; in fact, normal glues are based on polymer bridges whose structure is poorly controlled, thus making impossible to carefully modulate, preferably, on a molecular basis, the mechanical properties of the adhesion; (ii) strong adhesion of different and/or non- adhesive materials, like, graphite, teflon, silicon, copper, silver, palladium, platinum, mercury;
(iii) non-immunogenic adhesion, in particular, when applications in bio-medicine and biotechnology are needed, or in any other contexts where the materials could come in contact with persons, e.g. on the surface of objects that can be directly handled by humans; (iv) adhesion in aqueous conditions:, it is known that conventional glues most often require organic solvents and the very strict exclusion of water traces; (v) miniaturization of the gluing process, so that either microscopic objects (for example, cells' or the like, biological tissues and so on) or macroscopic objects (for example, surfaces of inorganic or organic objects) can be properly • ■ ' glued; furthermore, the glue can be used on micro-patterned macroscopic objects, to select on said objects areas to be glued 'and areas not to be glued, for example, on microscopic 5 objects; or to better define fracture dynamics during the taking-apart of glued macroscopic objects, if desired;
(vi) reversible adhesion, which can be useful in biomedical applications or, in particular, in 0 the electronic industry.
The solution to the above problems would -have a great societal and economic impact, also of a long-term nature. Adhesion technology is very important, from all points of view, for a lot of fields of 5 application and would greatly profit from new and original approaches which are water-applicable, tunable on a molecular basis, miniaturizable, more selective in their function, more versatile and effective, as far as bulk. chemicals are involved. 0 On consequence, there is still a long felt need for substantial technical improvements in the field of adhesion technology.
In particular, there is a need for a gluing system and method that consent: 5 - a complete mechanical control, modulable on a molecular basis, along the whole extension of separation of the glued surfaces; ■ - 'strong adhesion of' different and/or non-adhesive materials; a non-immunogenic adhesion;
- adhesion in aqueous conditions; - miniaturization of the gluing process, so that either microscopic or macroscopic objects are glued;
- possible, reversible adhesion.
It is one aim of the present invention giving a proper response to the above described need.
This aim, and others which will clearly result from the following description, have been reached by the Applicant, who has unexpectedly found that it is possible to give a proper response to the above described need by using DNA, or fragments or analogs thereof, as a molecular glue for gluing microscopic or macroscopic objects.
Accordingly, it is an object of the present invention the use of DNA, or. fragments or analogs thereof, as a molecular glue, " as disclosed in the appended independent claim.
It is another object of the present invention the use of DNA, or fragments or analogs thereof, for preparing "' a molecular glue, as disclosed in the appended independent claim. '■ "•
It is a further object of the present invention a • method for gluing objects by using DNA, or fragments or analogs thereof, as disclosed in the appended independent claim.
Preferred realizations of the present invention are disclosed in the appended dependent claims. Further objects of the present invention comprise a DNA based glue' composition and a method for preparing and using said composition.
The present invention is disclosed and detailed in the following description. Additionally, the invention is better clarified with the help of the enclosed Figures from 1 to 3. wherein: Figure 1 (Fig.l) discloses the basic concept of the DNA glue approach, wherein two substrates (micro or macroscopic) are, firstly, functionalized with two different sets of complementary chains of ODNs and then brought in contact to each other. Fig. 1 shows that the more the two ODNs are complementary (in this case, they are fully complementary) , the more strongly they will hybridize and more strongly the two substrates will be glued together.
Figure 2 (Fig.2) diβcloses: a), a DNA adhesion bridge; b) , "the mechanical opening of the DNA bridge in a. shearing topology (this is a non-equilibrium instant event, as revealed by a saw-tooth pattern in its single-molecule-force-spectroscopy spectrum, reporting the force vs. the extension while the two surfaces are pulled apart —• FSH is the force at which the bond breacks; c) & d) , strengthening the adhesion bridge by adding a TFO to form a triple helix [c) ] or psoralens to add covalent cross-iinks between two hybridized DNA strands [d) ] . Figure 3 (Fig.3) discloses: [a), c) , d) ] , DNA adhesion threads with sacrificial structures to cushion stresses on the contacts of the two surfaces and to modulate and maintain their interaction over great extension their interaction; b) , the unzipping of intra chain loops (this is an equilibrium reversible process that takes place at a constant force, F0Z/ as revealed by force vs extension measurements on a single molecule) ; e) , strengthening the sacrificial structures and the adhesion bridge shearing off.
DESCRIPTION OF THE INVENTION
The present invention is directed to the use of DNA or fragments or analogs thereof as a molecular glue for gluing micro and macroscopic objects/substrates on a molecular basis.
Preferably, said DNA or fragments or analogs thereof are selected from: natural or synthetic DNA or pieces thereof, of ' different length, single strands of DNA (ssDNA) , natural . or synthetic oligonucleotides, chains of different length of oligodeoxynucleotides (ODNs) , triple-helix-forming oligonucleotides (TFOs) , or analogues thereof; hybrid structures of DNA, ssDNA, or ODNS, or analogues thereof, with synthetic or natural polymers, like, for example, polystyrene', polyamides, polypeptides, silicones . and so on.
Preferably, they are selected from ssDNA or ODNs or analogues thereof. Most preferably, they are selected from ODNs.
Preferably, said objects/substrates are selected from: non-adhesive materials, like, graphite, teflon, silicon; - inorganic , materials,. like, copper, silver, palladium, platinum, mercury; organic materials, like, natural or synthetic polymers, like polydimethylsiloxane; biological materials, like, -proteinaceous or carbohydrate-based materials.
Preferably, said objects are selected from: copper, silicon, graphite, polydimethylsiloxane.
Said objects can have any type of suitable shape and dimension, from the micro to the macro scale. The DNA based glue of the present invention is very useful for gluing, among the others, both flat or rough surfaces of different type of materials .
Just to mention a possible, absolutely non-limiting example, in a preferred realization of the present invention, one small copper plate of suitable form is strongly linked to a polymeric plastic surface to provide a component of an electric circuit.
The method of gluing objects with DNA or fragments or analogs thereof substantially comprises: bringing into contact to each other two objects/substrates as above- defined, wherein each substrate is functionalized with at least one single strand of said DNA, fragments or analogs thereo-f, and wherein each of said single strand functionalizing one of said substrates is at least partially complementary to one of the single strands functionalizing the other substrate; - letting hybridize said at least partially complementary single strands to give adhesion bridges between the two substrates; the number of said adhesion bridges is directly dependent on the grade of complementarity existing between the two single strands.
The higher the number of adhesion bridges, the stronger the bond realised between the two obj ects/substrates . The functionalization of said objects/substrates is carried out by first properly modifying DNA, or fragments or analogs thereof, with chemical residues that bind to the object to be glued, and then by letting react said modified DNA, or fragments or analogs thereof, with said object- to give the desired ■ functionalized substrate.
Said functionalization can be carried out, preferably, by using synthetic methods known in the art. Preferably, the hybridization phase of the above disclosed method is performed in aqueous conditions. Preferably, in the presence of a "hybridization buffer 1" comprising 80 mM MOPS (pH 6.8-7.5) and 100- 500 mM NaCl or of a "hybridization buffer 2" comprising 80-120 mM MgCl2.
In a procedure of general application, each surface of the materials to be glued together is prepared separately with known methods, accordingly to the type of material. For example, silver or copper surfaces are pre-cleaned (a variety of known methods can be employed) and then treated with thiol- derivatised ODNs or analogues thereof. This treatment is preferably made with as concentrated as possible derivatised ODNs aqueous solutions.
Said treatment is carried out for a time comprised from minutes up to days, depending on a .number of parameters (the type of material/s, its thickness, its dimension; the type of application and the type and strength • of the final gluing desired, for example, reversible or not) . Preferred are times as long as possible.
The two wet surfaces thus functionalized are then brought together and kept in contact until the desired gluing has been obtained.
Said surfaces and kept in contact at a temperature higher than 250C; preferably, higher than 500C; most preferably, comprised from about' 600C to 950C. In a preferred embodiment the temperature is comprised from about 70°C to about 95°C; most preferably, from about 800C to about 900C. Preferably, after heating temperature is lowered to room temperature (about 200C to 250C) or even to values lower than room temperature, while keeping the two surfaces in contact. Adhesion thus develops to the desired grade. The basic concept of the gluing method above described is exemplarily sketched in Figure 1 in its simplest approach, wherein two substrates are first functionalized with different deoxyoligonucleotides
(ODNs) and then are brought into contact to each other, to give adhesion between the two substrates trough hybridization of the complementary ODNs chains. The more the ODNs functionalizing the two substrates are complementary, the more strongly they will hybridize with each other and the more strongly the two substrates will be glued together. The mechanical resistance of the adhesion threads settled by the base-pairing code is controlled by the unzipping or, more commonly, by the shearing of the hybridized strands of the complementary DNA or ODNs chains (as disclosed in Fig. 2a) enclosed) . Shearing is an irreversible process with a force spectrum characterized by saw tooth profiles (as disclosed in Fig. 2b) enclosed) . The mechanical resistance of said ' • DNA adhesion threads can be improved by further adding, for example, during the hybridization phase, one or -more substances able to strengthen said resistance by forming additional bonds between the complementary DNA or ODNs chains.
Said additional substances are preferably selected among: triple-helix forming oligonucleotides (TFOs), any crosslinking agents, like, psoralene, or others, useful to said scope, suitably selected among the commercially available crosslinking agents.
In a preferred realization, the mechanical resistance of the same segments of hybridized strands above described is increased by the addition of a triple- helix forming oligonucleotide (TFO) or of a chemical crosslinker, like, for example, psoralene (as disclosed in Fig. 2c) and d) enclosed). In another preferred realization said additional substances can also be linked directly to one of the DNA or ODNs during the functionalization of one of the objects/substrates to be glued.
The force of shearing is dependent on the length of the paired ODNs and on the environment conditions: 50 pN can be considered a lower bound for the force necessary to shear, apart two short paired ODNs a few tens of base-pairs long.
Another mechanically relevant topology can be advantageously introduced in the DNA adhesion threads: that of intra-chain loops capable of "sacrificial" unzipping transition (as disclosed in Fig. 3a) enclosed) .
The 'sequence of a DNA chain makes it possible to tailor adhesion bridges between two surfaces with "sacrificial" superstructures that unfold and release the tension at forces lower than those requested to break the same adhesive bridges. The mechanical properties of the adhesion could be very precisely tuned in this fashion. The unzipping of two strands is an equilibrium process that requires a force much lower (Fπz = 9-20 pN, as disclosed in Fig 3b) enclosed) than that of their shearing (FSH = 50-70 pN, as disclosed in Fig. 2b) enclosed) . The unzipping topology can be variously combined with the shearing topology (as disclosed in Fig. 3c) & 3d) enclosed) . Because of the opening of these sacrificial loops at forces that are smaller than those required to shear-off the adhesive segments, the two glued surfaces will be pulled apart under external forces in a stepwise manner. This cushions stres.ses on the contacts of the two surfaces and modulates and maintains their interaction over great extension. As already mentioned, also the mechanical strength of these sacrificial structures can be further modulated by the addition of suitable TFOs and/or psoralene/s • (as disclosed in fig 3e) enclosed) . The design of the sequences for the self-assembling of the adhesion bridges is performed according to the object/substrates to be glued and to the needed strength and persistence of bonding. That consents to modulate gluing at the single molecule level obtaining the desired results of micronizing said gluing process and, at the same time, of controlling adhesion along the whole extension of separation of the glued surfaces. The control of said process at the molecular level is possible by employing, for example, the Force Spectroscopy methodology [as described in: X.Zhuang, M.Rief, Curr. Opin. Struct. Biol.2003, 13, 88-97]-.
A simple prediction and study of the adhesion- strength deriving from the simplest architecture has been made. The surface density of covalently attached oligonucleotides has been proven to fall easily in the range of 3-4 '1O12 molecules/cm2. Considering 1012 molecules/cm2 and assuming all mechanical shearing forces independent, mechanically coupled and happening at 50 pN (50»10~12. N),. such molecular architectures could join and hold together two 1 cm2 samples with 50 N force in water (practically hold a
5 kg weight, or two 10-by-lO cm samples with a 5000 N 'force) .' This results is valid assuming that complete base paring between molecules on both faces of the glued objects happens. In case simple base pairing happens (i.. e. with no cross-linking, or formation of triple helixes) , that implies that such a strong bond should be completely reversible (for example, by raising the temperature, or by using proper nucleases, in a biological environment) . •
Less reversible networks obtained by covalent cross- linking, or other strengthening strategies as above disclosed, can take the force for separating two paired strand of ODNs up to the nanoNewton regime. Using the same parameters and assumptions of the previous calculation, the rupture force for DNA bridges covalently sealed after the hybridization can result of around 1O12»1CT9 = 103 N for a sample of 1 cm2. These simple calculations show the usefulness of a DNA based self-assembling glue.
The DNA gluing method of the present invention is advantageously used for selectively gluing on a molecular basis either microscopic or macroscopic objects as previously described. Said method resulted particularly advantageous for gluing in a very specific way object, like silicon and graphite, that can be ■ prepared atomically flat.
This requirement resulted very useful for the evaluation of the adhesive properties of the DNA glues without the influence of the surface roughness.
However, the method resulted highly promising also for gluing rough surfaces. Various methods are available to tether nucleic acids to a large variety of surfaces, including copper, silver, palladium, platinum and silicon (through amino-silanes treatments and homobifunctional or heterobifunctional crosslinkers selected from the ones commercially available) .
The attachment of ODNs to a graphite surface is very- difficult, because said functionalization may lead to the destruction of the flatness of the graphite structure.' However, the functionalization with ODNs of large molecules, like, for example, the grapitic derivative hexa-peri-hexabenzocoronene was able to give acceptable results. The ODNs are synthesized using ' standard phosphoramidite chemistry for preparative ' preparations. For example, in a realization of the invention, operating an ODN synthesizer (for example a "Gene-assembler" , from Pharmacia) in two scales, 150μmol and 30μmol, it was possible to obtain the desired amount of a 20mer (20 nucleotides per chain) , corresponding to around 100 and 20 mg, respectively. These quantities allowed gluing together large surface areas' of macroscopic objects. The following structural features (relevant to an efficient gluing) were screened on interplaying their empirical variation with their tailoring on the basis of force measurements at the bulk and single molecule level: (i) number of base pairs per oligonucleotide and degree of match/mismatch in hybridisation;
- (ii) nature and length of the spacer;
(iii) nature of the anchor group linking the substrate to the ODN;
- (iv) loading density (i.e. amount and concentration of the ODN solution per area of substrate) .
Where desired, in order to get more strength of bonding, in particular, in the case of biological applications, the ODN has " been modified ' to get resistance to nucleases, following the strategies developed for the antisense technology.
In another possible application, nucleases can ensure another way of reversibility of the gluing process for biomedical applications. The slow rate of this process can open further possibilities for that applications based on an adhesion that must be slowly released with time
The mechanical properties of the different DNA constructions tailored for the adhesion threads have been studied at the single molecule level, by the employing the Dynamic Force Spectroscopy methodology, the tool of choice to probe relationships between rupture ■ force, lifetime and chemistry in single molecules bound in an adhesion complex, and to reveal details of the adhesion energy landscapes at the molecular scale.
The evaluation of the resulting mechanical properties at the macroscale fracture mechanics testing, and the investigation of the continuity between the mechanical properties at the micro and at the macro- scales by analysis of the micro-mechanisms of adhesive fracture has given very promising results.
Moreover, thanks to the possibility .of modulate the synthesis of ODNs, by determining and using the most suitable sequences of bases, it is possible ■ to prepare (in case, on a case by case basis) biocompatible glues that can be used inside living organisms, for example, for biotechnological applications.
All that above shown confirms that DNA, or fragments or analogs thereof, can be advantageously employed as molecular glue. In particular, they are particularly useful for preparing glue compositions • for gluing micro and macroscopic object/substrates, that assure:
(i) the mechanical control along the whole extension of separation of the glued surfaces; the precision of the self assembling processes driven by. DNA fragments ensures an unprecedented possibility of modulating the structure of the adhesion bridges, thus finely tuning and tailoring the needed mechanical properties; (ϋ) the adhesion of non-adhesive materials, like graphite, as well as well known non-adhesive materials; conventional adhesives are unsatisfactory because they depend only on van der Waals forces, while DNA based adhesives act by controlling specific bonds between the surface molecules, thanks to the variety of functionalization chemistry available for ODNs, thereby increasing bond strength in a controllable and specific manner;
(iii) a non-immunogenic adhesions for applications in biomedicine and biotechnology; route towards glues that can even be degraded over a limited time, if inside the body of a living organism, without posing health dangers;
(iv) the adhesion in aqueous conditions; as DNA base pairing occurs in aqueous conditions, the gluing process using DNA as an adhesive also allows the presence of water, in contrast to conventional glues; (v) the possibility of reversible adhesions; when ssDNA strands have not been covalently crosslinked during the adhesion process, the DNA glue is thermoreversible and this would meet a" pressing need also of the electronics industry; (vi) the miniaturization of the gluing processes; by patterning the surfaces with different ODNs sequences, it is possible to auto-assemble various microscopic . or macroscopic objects, also compartmentalizing the gluing process; (vii) the adhesion to recognised objects; at present, adhesives are non-specific and attack, or try to attack, to any objects in their contact, while, on the contrary, the DNA adhesive is extremely specific and gives good adhesion when the correct molecules are available at the surface.

Claims

1. Use of a DNA, or a fragment or an -analog thereof, as a molecular glue for gluing microscopic or macroscopic objects/substrates.
2. The use according to claim 1, in which said DNA, or fragments or analogs thereof, are selected from: natural or synthetic DNA or pieces thereof, single strands of DNA, natural or synthetic oligonucleotides, chains of oligodeoxynucleotides, triple-helix-forming oligonucleotides, or analogues thereof; hybrid structures of DNA, ssDNA, ODNS or analogues thereof with synthetic or natural polymers. ■
3. The use according to claim 2, in which said DNA, or fragments or analogs thereof, are selected from ssDNA or- ODNs or TFOs or analogues thereof.
4. The use according to claim 3, in which said DNA, or fragments or analogs • thereof, are selected from ODNs.
5. The use according to claim 1, in which said objects/substrates are selected from:
• ' - non-adhesive materials, ■ like, graphite', teflon, silicon; - inorganic materials, like, copper, ' silver, palladium, platinum; organic materials, . like, 'natural or synthetic polymers, like, polydimethylsiloxane; biological materials, like, proteinaceous or 'carbohydrate-based materials.
6. The use according to claim 5, in which said objects/substrates are selected from; copper, silicon, graphite, polydimethylsiloxane.
7. The use according to claims 5 and 6, in which said objects /substrates are flat or rough surfaced.
8. .A method for gluing objects by using DNA, or fragments or analogs thereof, comprising: - bringing in contact to each other two objects/substrates as described in anyone of claims from 5 to 7, wherein each one of said objects/substrates is functionalized with at least one single strand of said DNA, fragments or analogs thereof, and wherein each one of said single strand functionalizing one of said objects/substrates is at least partially complementary to one of the single strands functionalizing the other substrate; letting hybridize said at least partially complementary single strains to give adhesion bridges between the two substrates.
9. The method according to claim 8, in which said objects/substrates are functionalized by:
-firstly, modifying said DNA, or . fragments or analogs thereof, with chemical residues that bind to the object to be glued; and
- then, letting react said modified DNA, or fragments or analogs thereof, with said object to give the functionalized substrate.
10. The method according . to claim 8, in which said hybridization phase is performed in aqueous conditions .
11. The use of DNA, or fragments or analogs thereof, as described in claims from 2 to 4, for preparing a molecular glue for gluing microscopic or macroscopic objects .
12. The use according to claim 11, for applications in biomedicine and/or biotechnology and/or in the electronic and/or automotive industry.
PCT/IB2006/000275 2006-02-01 2006-02-01 Use of dna or fragments or analogs thereof as a molecular glue WO2007088406A1 (en)

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Citations (2)

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DE10010118A1 (en) * 2000-03-03 2001-09-20 Henkel Kgaa Coating system, useful for the production of microelectronics, comprises a pyranosyl nucleic acid comprises a pyranosephosphate back bone bonded to purine or pyrimidine bases.

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WO2001034765A1 (en) * 1999-11-08 2001-05-17 Nanogen, Inc. Methods and apparatus for the electronic, homogeneous assembly and fabrication of devices
DE10010118A1 (en) * 2000-03-03 2001-09-20 Henkel Kgaa Coating system, useful for the production of microelectronics, comprises a pyranosyl nucleic acid comprises a pyranosephosphate back bone bonded to purine or pyrimidine bases.

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