KR20110081139A - Single strand dimensional construction of dna in 3d space - Google Patents

Abstract

The process of generating a three dimensional (3D) addressable array of biopolymer nucleic acids comprises providing a nanoscale three dimensional shape with a surface and combining the plurality of biopolymer nucleic acids in a regular pattern on the surface of the 3D shape. Steps. 3D biopolymer nucleic acids can be hybridized with complementary probes with reactive chemical groups to provide enzymatic active site simulations or artificial catalysts. Such simulations or catalysts can be used in biofuels and other industries.

Description

Single Strand Dimension of DNA in 3D Space {SINGLE STRAND DIMENSIONAL CONSTRUCTION OF DNA IN 3D SPACE}

The present invention relates to a three-dimensional addressable array of biopolymeric nucleic acids and to a method of making such arrays. Such arrays can be functionalized with complementary chemical reactive probes to provide catalytic enzymes.

This application is incorporated herein by reference in the following applications: US Provisional Application No. 60 / 918,144, filed Mar. 15, 2007; US Provisional Application No. 60 / 969,154, filed August 30, 2007; US Provisional Application No. 60 / 985,961, filed November 6, 2007; US Application No. 11 / 936,045, filed November 6, 2007; US Provisional Application No. 61 / -032,118, filed February 28, 2008; PCT Application PCT / US08 / 57013, filed March 14, 2008; US Provisional Application No. 61 / 043,981, filed April 10, 2008; US Provisional Application No. 61 / 047,201, filed April 23, 2008; US Provisional Application No. 61 / 048,599, filed April 29, 2008. References cited herein may be helpful in understanding the invention and are each incorporated herein by reference.

It is an object of the present invention to provide a three-dimensional addressable array of biopolymeric nucleic acids and a method of making such arrays.

The present invention relates to a method for generating addressable arrays of biopolymeric nucleic acids linked to three dimensional (3D) substrates that can be used as nanoscale fabrication platforms. Biopolymeric nucleic acids may include DNA, PNA, RNA, or LNA. Biopolymer nucleic acids can be used in dip pen nanolithography, femtosecond laser-mediated patterning through a sacrificial layer, laser conditioning on grooves or risers previously formed on the surface. It can be bonded to the substrate using lithography, or a magnetically aligned alignment over a contoured surface. Biopolymer nucleic acids may be attached to metal (eg gold), plastic and / or polymeric substrates. Various molecules, such as amino acids, can be linked to 3D addressable platforms. Activated platforms can be used as catalysts or as enzyme active site mimics. Platforms can be used to generate new catalysts through combinatorial chemistry.

The invention further relates to a method of replicating addressable arrays of biopolymeric nucleic acids linked to 3D substrates using impression molding, wherein the replicated array is transferred to a flat stamp or roller. . Alternatively, the replication method may comprise the use of compression molding, in which pressure is added to an elastic material consisting of a bladder, or the elastic material is changed to an environment (reagent, temperature). Etc.) to expand to interact with the array being replicated.

The accompanying drawings, which are incorporated herein and form a part of this application, together with the description, present the invention and describe the invention. In the drawings, like elements are referred to by like numerals.
FIG. 1A is a schematic diagram of cellulase progressing with respect to cellulose microfibril. FIG. FIG. 1B is a schematic representation of a synthetic glycoside hydrolase (E1 Endoglucanse mimetic) platform axially arrayed with ssDNA and hybridized with functionalized probs to be. 1B also shows cellulose oligomers for size comparison.
FIG. 2 is a master, such as a representative active enzyme site that can be prepared by nanoscale lithography, including a cylindrical, sharp (point) gutter, cleft / crater and compound helix. A schematic drawing of the master shapes.
3A is a schematic diagram of ssDNA arranged in regular 2D patterns. FIG. 3B is a schematic representation of ssDNA lithographically atop a 3D univariable radius mold. Also shown are electrical leads used to manage the electrostatic potential of the gold template unit.
4 is a schematic diagram of an ssDC-specific change in lift-off generation.
5 is a schematic diagram of representative molds used for templates and platforms of NIL system generation. 5A is a roller-type array of hemispherical shaped molds. 5B is cut cone shaped molds of a flat stamped array.
6 is a schematic diagram of 3D platform generation from templates.
7 is a schematic diagram of 3D template generation from masters using an elastic template material.
8 is a schematic diagram of 3D template generation from a master using an inflation template material.
9 is a schematic representation of a representative ssDC catalyst.
10 is a schematic diagram of an assembly of universal catalyst platforms having helical and gap geometric shapes.
FIG. 11 is a schematic diagram of a representative ssDC ™ “Catalyst-on-a-Chip:”.
12A-C are representative diagrams that enable techniques for cybernetization.
FIG. 13 is a schematic representation of twenty natural occurring amino acids, representing a majority of monomeric biological enzymes.
14A-C are schematic diagrams of catalysis within the present application.
FIG. 15 is a schematic diagram of an exemplary master shape including permanent ssDNA lithographically lithographically patterned in a pattern on gold.
FIG. 16 is a schematic diagram of ssDNA repeatedly fixed on DPN-spot type receptor points and unfolded linearly across template shape to produce Cartesian patterns.
FIG. 17 is repeatedly fixed on Fsec laser-depleted points through functionalization with SA or anti-DIG on a sacrificial layer and spread linearly across the master shape. Schematic drawing of ssDNA.
FIG. 18 is a schematic illustration of an ssDNA curved or linearly unfolded in pre-lithographed grooves repeatedly fixed on Fsec laser-consuming points on a 2D surface and a 3D master contour. to be.
FIG. 19 is a schematic illustration of one ssDNA strand unfolding from a Fsec laser-lithographically fixed point into a confining tunnel where it is changed into a groove or rise through a linear vector magnetic field line.
FIG. 20 is a schematic diagram showing the unfolding of the ssDNA component modified with phosphorothioate on lithographic gold through unwinding and unfolding from the colloidal enclosure affected by the movement of the magnetic field.

Part  Ⅰ. Single component dimensional structure

Single component dimensional structure (also referred to herein as "ssDC"; both "single component dimensional structure" and "ssDC" are trademarks of lncitor LLC) are physical-chemical in three-dimensional space Single component DNA and nanoscale forms can be used to address phosphorus functionality, and the development and generation of nanoscale 3D structures may require specific batches of discrete molecules to provide catalysts and other products. . Heterogeneous catalysis, polymer dynamics, multifacial organic synthesis, supramolecular assembly, microarrays and directed evolution / combination chemistry (DE / CC) By combining aspects of ssDC can enable mass production of various products. Examples include potentials for replication and amelioration, mostly depending on the existing enzymes and findings of new modes of catalysis.

ssDC is based on a single component template preparation method (sometimes referred to herein as "SSTM"; both "SSTM" and "single component template preparation" are trademarks of lncitor LLC) and mimic peptides ( mimetic peptides), biopolymers, and other molecular structures directly from templated DNA can be synthesized. With SSTM, high density arrays of DNA addresses are anchored to two-dimensional (2D) surfaces with geometric precision and serve as fabrication platforms, on which the composites are combined with DNA in the solid phase. In bulk, this is facilitated through coupling chemistry and hybridization which occur through stable interfaces between monomeric components polymerized in the liquid phase.

Since the fabrication platform shape of ssDC is not limited to 2D (or “flat”) geometric shapes and is not the end products needed to be disconnected from the template after synthesis, ssDC is capable of reducing the functional groups under conditions where extra dimensions are beneficial. It may also indicate a viable choice. As a result, 3D platforms are a bottom-to-top assembly of supramolecular products with many products and processes, e.g., stereo-specifically placed components. It can serve as a basis for multifacial synthesis facilitated by catalytic centers that perform functions such as, and enzymes.

A third application of this example is the generation of platforms similar to enzymes or bio-inspired heterogeneous catalysts will be described in detail below. The techniques and methods described herein are meant to be representative only of the overall technology and its application.

Examples using ssDC as a process for generating catalysts

An example of the application of the present invention is to develop catalyst platforms and production templates with geometric shapes produced by enzymes useful in the biofuel industry. Most cellulase and other glycoside hydrolases largely carry out their functional groups in gap-like active sites, where the gap-like active sites recognize, bind and locate substrates, stabilize and promote the formation of intermediates. And generate and release products that are generally fermentable sugars (see GJ Davies and B. Henrissat, "Structures and mechanisms of glycosyl hydrolases," Structure 3 , 853 (1995)). The present invention seeks to enable successful structures of nanoscale catalysts that may be similar to these geometric shapes and functionality, while also using more elastic materials in their structures.

1 illustrates the concept. 1A shows cellulase progression over cellulose microfibrils (public domain image of the US Dept. of Energy, Oak Ridge National Laboratory, ORNL Review 40 (1), (2007)). 1B shows a synthetic glycoside hydrolase (E1 endoglucan seminetic) platform axially arranged in ssDNA and hybridized with functionalized probu. Cellulose oligomers are shown on the platform for size comparison.

A method for generating a catalyst using ssDC includes the following steps: a) generating a shape having nanoscale dimensions similar to the geometric shape of the enzyme active and / or binding sites; b) binding the ssDNA to the nano shapes in a repeatable and predictable manner; c) selectively regenerating such ssDNA impregnated nanoshapes using large amounts of production techniques; And d) functionalizing the ssDNA-impregnated nanoforms by binding complementary ssDNA sequences bound to various molecules, such as amino acids, at specific positions on the nanoforms.

For the purposes of this description, the ssDNA impregnated nanoforms from steps a) and b) are referred to as "masters." Mirror images of these masters are known as "templates." Accurate reproducibility of masters functionalized with ssDNA sequences bound to various molecules of interest is known as “platforms”. As can be evident, masters may be platforms if the master eventually becomes functional.

The term "polynucleobases" or "biopolymeric nucleic acids" refers to single component DNA, monocomponent RNA, PNA, or LNA, heterogeneous monomeric (ie homogeneous monomer) Copolymers of deoxyribonucleotides and nucleobasic beta-amino acids, which leave about 0.5 nm space and maintain the stacking capacity of the bases). As an example of the present invention, the indication “ssDNA” will be used herein to refer to the above biopolymer nucleic acids modified for permanent attachment on masters, and will be used as complementary biopolymers in lithography of templates.

Creation of masters

The process of creating masters begins with a 3D nano-shaped, concave or convex state. Such nanoforms can be generated by a number of different methods, including but not limited to electron-beam lithography, nanoimprint lithography, and / or deep pen nanolithography. . For the purposes of this example, 3D pattern transfer nanoimprint lithography (see Kim et al., "Three-dimensional pattern transfer and nanolithography: modified soft molding", Appl . Phvs . Lett . 81 , 1011 (2002)), or rigid- By selective removal of the sacrificial layers deposited on the hard-cast material, light- or electron-beam lithography that can produce arrays of accurate mold shape, size and separation distance (+/- 10 nm) Gap-shaped depressions generated by the procedure (see SY Chou et al., “Sub 10 nm Imprint Lithography and Applications”, J. Vac . Sci . Technol . B 15 (6) , 2897 (1997)) are considered. The process can be completed without all the steps described herein (e.g., the catalysts can be constructed directly from the functionalized masters) and reversed for example to start from convex rather than concave shape. Can be. In determining the precise shape and dimensions of the gap, measurements from the activity and / or binding site (s) of the enzyme can be used, computer generated models of the active site of the enzyme can be used, or the shape is only readily available. Can be determined. The end product version of the shape can be functionalized into physico-chemical groups to mimic catalysis. Arrays of these same shapes can then be fabricated on a surface, such as Si (100) or Si0 ₂ (two many examples) or other material, by light- or electron-beam lithography, and by other methods, The quality is then inspected by atomic force microscopy (AFM) in tapping mode, scanning electron microscopy or other microscope that can see the nanoscale to demonstrate geometry.

FIG. 2 shows a master shape, such as a representative active enzyme site that can be prepared by nanoscale lithography, including a cylinder 22, a sharp (point) trough 24, a gap / hole 26 and a compound helix 28. Schematic drawing.

If an array of identical shapes are synthesized on a single surface, one of the two processes may vary depending on the process used to functionalize the final catalysts: 1) The influence of these master shapes is low intrinsic viscosity and small bubble size space Can be taken through small bleb size space-filling polymers (see GH Cross et al., "Refractometric discrimination of void-space filling and swelling during vapour sorption in polymer films," Analyst 125 , 2173 (2000)). . After casting, polymerization can be initiated by UV light or other methods. Such rigid-cast permanent molds can be used to reverse the creation of additional masters, or end-user platforms of mass production; Or 2) ssDNA can be added to the shapes of the array, and the shapes of the array are referred to as the "master".

The first procedure can be used when the production of catalysts requires induction of separation of the form followed by addition of DNA. A master without ssDNA is used to derive the shape. The master with ssDNA then adds DNA to the shape.

The second procedure is used when the shape and the DNA are applied simultaneously in the preparation of the catalyst. While the second process of adding ssDNA to the master is described more fully in Part III of this description, some of the available options for adding ssDNA are: (i) deep pen lithography (nanoscale stylus And alignment of the DNA through the influence of the interface), (ii) laser conditioning pattern lithography on pre-formed grooves or elevations that reduce translocational uncertainty, and / or (iii) adhesion to the ends of the DNA. Magnetic field patterning of unfolded DNA using linear portions of fields that affect the acceptable mass. Synthetic DNA can be chemically modified to form covalent bonds with the template and added in close proximity (10 nm distance) to pattern the high density addressable array on the final product. The resulting pattern can be curved or Cartesian to facilitate predictable addressing and simplify dynamic modeling of physico-chemical functions and catalysis profiles.

3 shows examples of ssDC specific changes to the basic patterning approach described in the referenced publication. 3A shows ssDNAs 31, 32, 33 and 34 arranged in constant 2D patterns (LM Demers et al., “Direct Patterning of Modified Oligonucleotides on Metals and Insulators by Dip-Pen Nanolithography,” Science 296 , 1836). (2002)). FIG. 3B is a schematic illustration of ssDNA 35 lithographically atop a 3D univariable radius mold (e.g., Au 36 disposed on titanium 37) (CM Waits et al., "Microfabrication of 3D silicon MEMS structures using gray-scale lithography and deep reactive ion etching, " Sensors and Actuators A 119 , 245 (2005)). Electrical leads 38 and 39 can be used to manage the potential of the gold template unit, which is also shown.

Once the masters are structured, the masters can be used in one of three ways, where the three methods are as follows: 1) If the masters do not have ssDNA on them, the masters are used to regenerate the nanoshape. Can be used; 2) if the masters have ssDNA on them, the masters can be used to regenerate a grid (and possibly shape) of ssDNA for the shape produced by the non-ssDNA active master; Or 3) if the masters have ssDNA on them, the masters may have complementary probes connected to the master that functionalize the master and convert it to a "platform", or functionalized ssDC product.

There are a number of ways to use masters to generate templates and end platforms, which can speed up production and lower production costs as desired. Examples of such methods include contact molding and pressure molding, each of which is described in detail below. These terms are not meant to imply a process existing outside of this description using such terms. The following parts describe "contact compression" and "compression molding" techniques that generate templates and platforms.

Contact Pressure Molding

In contact pressure forming, two different masters are used. The first consists of a master with geometry only. This master is imprinted on the soft polymeric material, the polymer is cured by UV light or other methods, the template is removed by lift-off and generates the original master shape in a dimensioned amount.

FIG. 4 illustrates lift-off generation, including ssDC-specific changes to the basic themes described in the reference publication (PW Snyder et al., “Biocatalytic Microcontact Printing,” J. Org . Chem . 72 , 7459). 2007); and Duke University Office of News and Communications, Sept. 2007). The template 41 with shaped shapes 42 is mixed with address nucleotides 43, and then imprinted on the polymer platform 44, and the probe ssDNA within the impressions. Eject 45, and maintain mirror-image complementarity addresses.

ssDNA can be added to these shaped templates by using a master with ssDNA attached to the master. Complementary DNA or PNA (peptide nucleic acid, Peptide Nucleic Acid) intended to form an addressable array of templates can be hybridized to ssDNA and master, and later "inking" the pattern shift. The nucleobase sequence complementarity and the mirror image of the DNA located on the master can be the amount generated as templates.

In most cases, templates can be generated from roller-like templates, or by stamp-and-peel molds, by rearranging empty templates with a master with ssDNA. 5 shows representative molds that can be used for templates and platforms of NIL system generation using ssDC-specific variations on the basic themes described in the referenced publication. 5A is a roller-type array of hemispherical shaped molds. 5B is cut cone shaped molds of a flat stamped array. 5A shows a roller array of hemispherical shaped molds (see H. Tan et al., “Roller nanoimprint lithography,” J. Vac . Sci . Technol . B 16 (6) , 3926 (1998)). 5B are cut cone shaped molds of a flat stamped array (S. Diegoli et al., “Engineering nanostructures at surfaces using nanolithography,” Proc . Inst . Mech . Eng . G: J. Aerospace Eng . 221 (4) , 589 (2007)).

In order to facilitate the transfer of template ssDNA or PNA, and to maintain the reusability of masters, lithographically ssDNA is for example via phosphorothioate-on-gold adhesion, or siloxane functionalized rigid casting. hard cast) may be covalently bonded to the master material via amine ssDNA backbones on Si (100). In addition, the migration of the polymer onto the template (ie, "raising nucleobases") in a manner that preserves the mirror image pattern and addressability of the template is as follows: change in temperature, master surface energy and potential, UV Or by other polymerization methods, roll to lift-off template, solvation, lubrication, mechanical and other elements. Platforms can be configured in the same way as templates, but can be configured using templates with or without ssDNA instead of masters with or without ssDNA.

6 shows an ssDNA migration method for both 3D templates and platforms. The figure shows a phosphorothioate-backboned ssDNA (ssDNA) 62 lithographically lithiated on a gold template 64 after being lifted off from platform 66, wherein the complementary address DNA or PNA 68 will remain.

Compression molding

Another exemplary method for regenerating a master in templates and / or platforms also uses a soft polymer or other material that is elastic or expandable under certain conditions. The master may be located in a container that defines lateral movement. The intended complementary DNA or PNA to form an addressable array of templates can be hybridized to ssDNA and master and later "inked" the pattern shift.

Thereafter, the soft polymer, or other elastic or expanded material, can be added to the container. If the material is elastic, the elastic material may be configured as a bladder. The outer surface of the bladder may be prepared to permanently bind to ssDNA. Pressure may be applied to the interior of the elastic bladder, which allows the material to expand to the dimensions of the container, adhere the shape of the master, and pick up complementary DNA previously hybridized with the master. up). The elastic material can then be cured in a manner suitable for its inherent properties, for example UV curing or other processes. When the material expands, instead of applying pressure, the conditions causing expansion (e.g., temperature change, pH change, etc.) can be introduced into the material such that it adheres to the shape of the master and obtains ssDNA. The same steps can be obtained and saved.

7 and 8 demonstrate these methods. 7 illustrates 3D template generation from a master using an elastic bladder. Due to the pressure, the template elastic material expands, takes the shape of the master, and connects to the complementary ssDNA previously laid down on the master. After the template is linked to the complementary ssDNA, the template can be peeled off. 8 shows 3D template generation from a master using expanded material. Due to environmental changes, the template expanding material expands, takes the shape of the master, and connects to the complementary ssDNA underlying on the master. The template can then be peeled off.

The manufacture of the platforms can proceed in a similar manner as the template manufacture. Templates can be used at the master's location for the structure of the platforms. These processes can be repeated as many times as necessary to generate the required number of platforms.

Activation of Platforms on Catalyst

ssDC platforms can be considered as nanoscale 3D high density addressable arrays with patterned DNA or PNA with nanometer-level precision capable of replicating billions of copies. The shape of the platforms may partially convert catalysts, such as enzymes, because they may resemble the size and shape of the active sites and can be transferred to complementary probes whose addressable DNA possesses reactive chemical groups. Activating ". Depending on the hybridization, these groups may be located in platforms at locations similar to orthogonal side chains of active site amino acids that facilitate the catalysis of enzymes inherently similar to the production of master shapes.

In the description of the structural changes that occur during the catalysis process (functions provided by linkers on probes and chemical groups mixed with platform addresses), surface energy, tribology, solvation and other factors important to catalysis. The activated platform now binds clean cellulose or algic material, stabilizes the form of the transition states, catalyzes the form of the transition states, and natural cellulases (glycosidases). And ssDC products with the ability to release depolymerized sugars into monomers such as alginases (trans-esterases).

Additional advantages provided by the use of nanoimprint polymer lithography originally developed in the microprocessor industry include, for example, electrical, magnetic, and nanoscale actuators that move, vibrate and / or change the shape of certain parts of an activated platform. Selective integration of light, and other systems that facilitate active enzyme-site migration. Active enzyme site flexibility during the catalysis profile is an important part of substrate binding, transition state formation, product release and other activities, where nothing prevents effective catalysis, and the active site is referred to as a "static heterogeneous catalyst." (EZ Eisenmesser et al., "Enzyme Dynamics During Catalysis," Science 295 (5559) , 1520 (2002); M. Karplus and J. Kuriyan, "Molecular dynamics and protein function," Proc . Natl . Acad . Sci . 102 (19) , 6679 (2005); A. Kohen and JP Klinman, "Enzyme Catalysis: Beyond Classical Paradigms," Acc . Chem . Res . 31 , 397 (1998); and CL Tsou, "Active Site Flexibility in Enzyme Catalysis," Annal . NY Acad Sci . 864 , 1 (1998).

9 is a representative ssDC catalyst 92 presenting a simple activation of a master or platform configuration 66 with chimaeric amino acid-DNA probes 94 wherein the chimaeric amino acid-DNA Probes 94 may enable chemical functionality in 3D space and simplify friction with negatively-charged phosphorothioate (P) or neutral (N) backbones 96. .

Combinatorial Development via Directed and Random Evolution

Since the product DNA or PNA can be reused indefinitely by being permanently mounted on an elastic material, DE / CC can be implemented on platforms that produce billions of combinatorial changes in a given shape, and many different shapes can be used for beam lithography. It is possible by. For example, the simple platform shown in Table 1 allows for forty (40) unique address positions, including only eight (s8) different ssDNA components lithographically lithographically cross-hatch patterned. do. Each of these addresses can be hybridized with a small probe library of oligomers having only one of each of the twelve 12 chemical functional groups, and can only be linked at one location (eg, the end of 3). . This may increase the probability that some simple catalysts can be useful even though this simplest attempt at random development can produce different combinations of 40 ¹² or 1.68E + 19.

Table 1. Combination changes represented by 40-mer platform and one monofunctional, single linked 40 × 12-mer probe library for each address.

Leverage provided by more "random" deployments is further suggested by the fact that orthogonal chemical functionalities can be located on both ends of the PNA probe at the backbone or in multiple locations. In addition, several functional groups can be located on the same probe molecule (if self-reacting, the appropriate protecting groups must be part of the PNA design), and the types of linkers used to connect orthogonal chemicals to the probes It can be modified to facilitate flexibility and optimize catalysis. Even in the absence of orthogonal functional groups, the amide backbone of PNA, and the diester vertebrates of ssDNA and RNA, provide a given charge, charge density, and solvation, counterions, dipole moments, It can be modified synthetically to provide surface energy for portions of the platform that provide management of dynamic solvability / friction and other important features of catalysis.

On the other hand, attempts to produce catalysts similar to enzymes can be inspired by, but not limited to, active site design and bio-catalysis profiles. Rather than relying exclusively on the power of probability, more sensitive changes in the geometric and chemical themes exhibited by protein enzymes can be undertaken to optimize known catalytic processes, and are themselves scaffolded in 3D space and scaffolded based on a particular sequence of dynamically shifted amino acids (R. Chakrabarti et al., "Sequence optimization and designability of enzyme active sites," Proc . Natl . Acad . Sci. 102 (34) , 12035 (2005) Reference). Computer modeling of catalysis profiles from binding to release can be varied to enable the following representative improvements of ssDC ™ catalysts on protein enzymes: (i) Increase in substrate binding affinity (surface energy "Stickyness" to the receptor region through administration), (ii) the amino acids aspartate (Asp) and glutamate (GIu) are -1 / 4-glycoins in cellulose A stronger orthogonal acid position, which has already been determined to effect acid-catalyzed hydrolysis of Glycosidic adhesion, and / or (iii) not natural cellulase to speed up the overall reaction. Modification of platform geometry to enable metal cofactors that contributed to hydrolysis. Those skilled in the art will recognize many other choices made possible by the present invention.

Such an effort can be diligently obtained for design purposes, such that some end products, for example, are (i) stubbornly resisting or "stuck" on these products, and (ii) acidic. Because they do not want to have a substance such that they damage the substrate or each other and / or (iii) are stubbornly contaminated with metals. However, as mentioned, both random development of discovery mode (most of the products are rejected as a matter of expectation), and directed translocation are all initially derived from the enzyme model only through proven pathways. Although, but later, useful trans-biotic catalysis paths are found, more independently increasing evolves can be used repeatedly to produce improved products. . This process can develop and discover catalysts much faster than would be expected with the qualification of protein-based enzymes to maintain their inherent limitations regardless of genetic engineering, production, purification, testing, and these efforts.

Directed and Random Evolution, and Combinatorial Chemistry includes countless materials produced by the human body as part of "self vs. non-self" cognitive function. Thus, antibodies that bind to most antigens may be used in a concert similar to how the immune system successfully produces. For example, most protein antibody molecules retain most of the usual "Y" shape, but hypervariable regions that recognize foreign and familial materials are oriented (when encountering heterologous antigens). ) And random (if system damage is not immediately identified) can be induced to generate a degree of change in mole number (10 ²³ ) through the genetic code of combinatorial shuffling, wherein Hypervariable regions are programmed. Either way, it proves useful between any point of development and the DE / CC aspect of ssDC can be used to meet consumer needs in the shortest possible time.

Some parameters that can be used with ssDC ™ catalysts with varying combinations are:

Platform size, shape, surface deformation and materials

The pattern and density of arranged nuclear base addresses

Composition of high density addressable arrays: ssDNA, RNA, PNA, and mixtures thereof

Orthogonal chemical groups: amino acids, transcendent (eg organic) chemicals

The presence, absence and labiality of the protection group;

The location of the chemical functional group (s) on the probe: terminal, intervention, mixtures thereof

Single, multiple, or no functional groups on the probes to manage surface charge

Linker groups length, stiffness, size, dipole moment, hydrophilicity / phobicity

Probe backbones: phosphodiester, phosphorothioate, amines, hydrazides, etc.

Integration with electronic, magnetic and / or optical systems

Aqueous, organic, interfacial catalysis in trans-biotic conditions: extreme temperatures, pH, pl, ionic salts, metal ions, high shear forces and integration in the liquid, solid and colloidal phases It includes.

De Novo Catalysis Discovery Through Directed Deployment

In addition to gradual implementation of better "mimetic enzymes" for the biofuel market, the present invention allows the development of platform geometries that are geometrically tailored to the potential for performing a wide variety of catalytic processes without obtaining from specific biological precursors. . Such general catalyst platforms (UCPs) can be based on ssDC technology and consist of nuclear base probes (ssDNA, RNA, PNA, etc.) activated with chemical functional groups on and across these amino acids. (See DRW Hodgson and JM Sanderson, "The synthesis of peptides and proteins containing non-natural amino acids," Chem . Soc . Rev. 33 , 422 (2004)). This combination of large libraries of completely non-biological products can produce stronger and more effective catalysts repeatedly, where the catalysts operate under low temperatures and are newer and simpler than are possible with protein-based enzymes. Catalysis profiles are shown. This discovery process is referred to herein as "Physical Directed Evolution" (PDE).

Exemplary methods can use cone shapes with small DNA libraries and peripheral DNA components arranged in the longitudinal direction. 10 shows an assembly of UCPs with helical and gap geometric shapes: (a) an address ssDNA arranged in an inner material stamped with a conical mold; (b) individual UCP units before being "activated" with probe oligomers; (c) a library of oligomers (ssDNA, RNA, PNA) finally modified with active functional groups; (d) individual UCP units "activated" by hybridization with the final modified oligomers; (e) two UCP units activated in different ways by hybridization with dissimilar oligomers; (f) a finally activated UCP unit cut from the mold to maintain the helix as it is; And (g) a finally activated UCP unit cut from the mold to form a univariable radius gap.

New trans-biotic catalysis profile and system integration

With PDE, it generates 3D catalysts (shape, DNA patterns, linker techniques, chemical functionalities and other ideal parameters repeatedly listed above, qualifying 3D catalysts, 3D catalysts) Multiple rounds may provide even more improved products to examine, reconsider, and redesign them, but the profiles of catalysis and modes not known so far that are not possible with protein based enzymes In addition, the platform properties such as surface energy (to determine charge, solvation and friction), and morphology flowable by electromagnetic (EM) fields are purely biomolecules ( biological molecules can be processed in an impossible way.

As implied, linkers that attach reactive chemicals and other functional groups to probe oligomers are active when the linker reacts to the presence of a substrate, facilitates the generation of intermediates, and generates during catalysis profiles. It aims to mimic structural changes that occur in enzymes. The potential best way ssDC provides is to enable structural changes through electro-active polymer foundations, where the electro-active polymer foundations are subject to contact with a substrate, intermediate or product. It may be bent and relaxed, open and closed, and expand and contract.

For example, the platform may be made of conductive channels arranged in resonant piezoelectric crystals and arranged in polymer blocks made of a material having a form sensitive to a defined EM frequency. This may enable one or more portions of the mimic active site to vibrate upon sensing that the contact is implemented with the substrate (or to constantly vibrate in the default mode when considering a constant current). This can be initially established at frequency and duration such as catalytic residues of mimicked glycosidase and migration of active site moieties. Subsequent frequencies, amplitudes and geometric shapes can be varied to optimize the catalyst rates with industrial processes that are optimized for the purpose of integrating the catalyst. Based on the optimized methodology of generating electroactive polymer based microprocessors, the integration of ssDC catalysts in the feedback and control system enables structural change and real-time monitoring and helps identify the most promising candidates during process-based directed deployment discovery. Can give

11 shows a representative ssDC ™ “catalyst-on-chip”. 3D shapes can be optimized with DE / CC for functionality, position, linker, friction and geometric shapes. For example, a lead 112 for electrical input-output, electro-active harmonic actuators 114 underneath remaining blocks, and an optically connected center 116 are shown.

FIG. 12 illustrates enabling techniques for cybernetization using ssDC-specific changes in the basic themes described in the referenced publication. FIG. 12A shows an array of multicomponents 121, 122, and 123 lithography on an orthogonal underlayer (LJ Guo, “Nanoimprint Lithography: Methods and Material Requirements,” Adv . Mater . 19 , 495 (FIG . And P. Choi et al., "Siloxane Copolymers for Nanoimprint Lithography," Adv . Func . Mater . 17 , 65 (2007)). 12B shows a nanoscale lens 124 made by double point (T, P) condensation of a polymer on a lithographic disk (P. Lenz and R. Lipowsky, "Morphological Transitions of Wetting Layers on Structured Surfaces," Phys . Rev. Lett . 80 (9) , 1920 (1998). 12C shows a detection system 127 that can provide an underlayer activation / monitoring system for off-axis nano-actuator / -resonator 125, signal buffer 126, and catalyst platforms (AM Fennimore). et al., "Rotational actuators based on carbon nanotubes," Nature 424 , 408 (2003)).

After being activated with functionalized probes, ssDC catalysts have the process advantages of hybrid catalysts, the combinatorial flexibility of reusable high-density addressable arrays, the strength of high impact industrial polymers and the biofuel monitoring process. Provides readiness integrated into Unlike biological enzymes, ssDC catalysts are not limited to orthogonal functional groups of amino acids, or platforms are not limited to a particular shape. They may be composed of substances that utilize conditions that would normally denature or neutralize biotic enzymes. Platforms can be easily integrated into industrial processes, such as packed filters, reaction columns, sedimentation materials and mixing surfaces. In addition, the incorporation of feedback / control systems enables monitoring of the catalysis process, easily identifies the most promising candidates for successive deployments, and speeds up development and optimization of new catalysts overall.

This effort can be obtained with process optimization and makes it possible to process designs for developing in-synch with the development catalyst system methodology. As a result, the entire biofuels production processes can be optimized at each stage to be implemented at peak efficiency no longer hampered by the restriction dots imposed using biotic enzymes. Higher or lower temperatures, as well as pH and ionic salt content, shear forces, organic solvents, and other conditions that optimize the hydrolysis and other biomass of cellulose (but they cannot currently be used, The reason is that they destroy protein-based enzymes) can be implemented with ssDC catalysts. Alternatively, ssDC catalysts can be developed to perform optimally for existing production processes.

SSTM as a stable interface that protects polynucleotide chains by immiscible aqueous / organic phase as long as the addressable nature of single component DNA is maintained (immiscible aqueous / organic phase). provided for ssDC, even the potential corrosive and corrosive organic reaction conditions of the bulk phase), combinatorial efforts can be functionalized for the desired product, single component DNA for site-specific locations It can be obtained to generate progressively improved versions of monomer components.

Once the ideal catalyst or other product is finished, additional steps can be started before system and process integration. Covalent bonds, such that de-hybridization is not possible, can be created between the probes and their addresses and permanently attach physico-chemical functional groups to the polymer based platform. If desired, this extra step increases the viscosity and usable life of the catalyst, particularly at high frequencies (e.g., higher than 1 gigahertz) required for dynamic catalysis to occur effectively, which is resonant and vibrating activity. Improve the ability of site functional blocks.

Finally, after the discovery based on combinatorial chemistry and deployment is complete, the inspection platforms can be “washed” back to simplify the 3D addressable platforms and hybridize with new sets of complementary probes. Preparation can be made. Then, they synthesize bio-mimetic polymers, discover other catalytic modes and profiles, perform supramolecular assembly, and bio-sensors, drugs It can be reused for a very wide range of purposes, including serving as high density addressable arrays for the construction of drug delivery vehicles, microchip components and other self-assembled devices.

Part  II. Heterogeneity  In the production of catalysts ssDC Application of

Process for developing significantly improved catalyst

The Single Strand Dimensional Construction (ssDC) technique described herein has many problems with protein-based enzymes, such as fragility, short shelf lives, limited catalytic function, and expensive infrastructure. Addresses production from growing genetically-engineered microorganisms. ssDC accomplishes this by handing off the production, development and function of biological enzymes to a nanotechnology-based platform that catalyzes in a more robust, versatile and active way.

Biological enzymes are made of polymerized amino acids. Twenty naturally occurring amino acids with different sequences, post-translational modifications and folded structures are responsible for catalyzing thousands of biochemical reactions. Although a versatile and effective construct that allows life to exist in a myriad of forms, enzymes generally have a slow rate of catalysis and are non-resistive to the environment, as are all naturally produced products. -recalcitrant) (ie bio-degradable).

In addition, the catalytic activity of enzymes is small, including acids, amines, amides and indole, "mild" electrophiles, and saturated and unsaturated hydrocarbons, all of which have limited reactivity and consequently limited catalytic strength. Derived from a set of chemical functional groups. The orthogonal potential of biological enzymes versus the "inferiority" of trans-biotic and artificial reagents are partially present, which means that proteins with too high reaction chemistry are sequestered in the cell. And are difficult to recycle, and are potentially toxic to both host organisms and the environment in general.

FIG. 13 depicts 20 naturally occurring amino acids and shows the monomer content of most biological enzymes (Department of Bacteria, University of Wisconsin-Madison).

ssDC technology can be used to make artificial enzymes, to develop new versions of these enzymes, and to discover new modes of catalyst in which natural enzymes cannot work. This is achieved by using a modified version of industry-standard nanolithography process in the construction of the platform on which the catalytic function is performed. The production and development methodology of ssDC is based on three-dimensional (3D) pattern transfer lithography. These versatile nanoscale level technologies can create platforms in a wide range of shapes and sizes, providing a basis for simulating any existing degrading enzyme.

Enzyme-Mediated Catalysis

The function of enzymes to promote biochemical reactions is highly dependent on the organization of protein structures with chemical functions that translocates inside the active site during the catalytic reaction. The site generally contains many dependent functional groups of the catalyst, including substrate binding, transition state formation and stabilization, chemical conversion and substrate release. As mentioned above, the binding of polymerized amino acids can facilitate thousands of different biochemical processes. However, the reaction generally takes place only in a limited temperature, pressure, salinity, ionicity and pH range, and in almost no exclusion of aqueous conditons.

The secret to the production of mimetic (ie, improved) versions of enzymes is to replicate the catalytic function on a more versatile, stable, and active platform. This is accomplished by first accurately modeling the active site of the enzyme into generalized lung curves, clefts and helices, and then fabricating this large array of shapes. Enclosures thus confine the substrate and place in the 3D space other amino-groups and other physico-chemical functionalities that contribute to the conversion of the substrate into products, such as electrostatic-based surface energy and dynamic conformational ability. It is designed to be.

The product of ssDC production is a heterogeneous catalyst in the solid state. However, these are the most advanced since their creation. Like fluidized bed reactors, reactive filters and biological immobilization enzymes, ssDC catalysts accelerate the conversion of liquid substrates into products through chemical functional groups immobilized on the solid side. As with most other heterogeneous catalysts, they perform best if the following are provided and optimized: The substrate dissolves well in the liquid state, generally brine, maximizes the surface area of the catalyst over the entire volume, and the catalyst surface Improved substrate transport (generally by mixing and / or phase separation) as substrates approach and products away, and the catalyst itself is designed correctly.

The "right" design of the catalyst allows a heterogeneous type of solid phase, such as an ssDC product, or a liquid / colloidal phase type with a subset of protein enzymes, to convert the substrate into a product in the shortest possible time and in the most energy efficient manner. It encloses an enclosed, active, highly specific chemical system. In the case of enzymes, this can be achieved by polymers of amino acids that are folded into a 3D structure. In the case of ssDC catalysts, this is achieved by using a platform that is formed like an enzyme active site and contains all the physico-chemical functionalities necessary to convert the substrate into a product.

FIG. 14 shows the catalyst in an enclosure that includes ssDC-specific variations for the base theme described in the referenced publication. 14A shows a simplified spatial filling model of the gap structure and functional groups common to glycoside hydrolase: acidic in spatial opposition with tyrosyl groups functioning as "positioners". And basic residues (W. Nerinckx et al., "An elaboration on the syn-ani proton donor concept of glycoside hydrolases: electrostatic stabilization of the transition state as a general strategy", FEBS Letters 579 , 302 (2005); And "How Family 26 Glycoside Hydrolases Orchestrate Catalysis on Different Polysaccharides" by EJ Taylor et al . , J. Biol . Chem . 280 (38) , 32761 (2005)). FIG. 14B shows zeolites designed as platinum atom traps (public domain image of the US Department of Energy, Berkeley Institute Research Report, Lawrence Berkeley National Laboratory , Fall 2001 ). FIG. 14C shows a 2D ring scaffold bound to supermolecules synthesized from cyclic alkene subunits ("Hterogeneous catalysis: what lies ahead in nanotechnology", Applied by HH Kung and MC Kung) Catalysis A: General 246 (2) , 183 (2003)).

PART  III. For catalyst development and production ssDC Of template  Options for Manufacturing

production

Use of ssDC to produce good catalyst

SsDC is applied to develop templates and fish platforms with structures inspired by the catalytic centers of enzymes useful in the biofuel industry. Most cellulase and other glycoside hydrolase perform their functions mainly in active sites such as gaps that recognize, bind and dispose of substrates, stabilize and promote the formation of intermediates, and release products. (DE Koshland, "Application of a Theory of Enzyme Specificity to Protein Synthesis", Proc . Natl . Acad . Sci . 44 (2) , 98 (1958); A. Warshel et al . "Electrostatic basis for enzyme catalysis", Chem . .... Rev 106 (8 ), 3210 (2006); West JB and the like of "Enzymes as Synthetic Catalysts: Mechanistic and Active-Site Considerations of Natural and Modified Chymotrypsin", J. Am Chem Soc 112, 5313 (1990) Reference). Successful nanoscale catalysts will mimic this structure and function, while at the same time utilizing more resilient materials within the structure (L. Jiang et al, "De Novo Computational Design of Retro-Aldol Enzymes," Science 319 ( 7) , 1387 (2008).

ssDNA Deployment Guidelines

In order to place the ssDNA in the master shape according to the present invention, the surface of the material can be functionalized for DNA lithography. In one example, physical deposition within the electrical potential between the solid and plasma phases is performed by titanium (250 A) for mercaptyl-to-gold anchoring of phosphorothioate modified ssDNA. It is possible to deposit a thin gold layer (50 A or less) having a back surface formed by the following). When using an amine backbone-modified ssDNA, the surface of the array utilizing the gapped texture from SiO ₂ can be chemically converted to siloxane modifiable by covalent bond formation to the amine. The method is designed to ensure backbone-specific bonding due to the presence of primary amines and guanidinium groups in the adenosyl, cytosyl and guanosyl systems. It requires more sophisticated functionalization. The master can then be ligraphed with ssDNA, RNA or PNA. DNA is synthesized and chemically modified to form covalent bonds with the master, and high density (10-25 nm spacing) to serve as a complementary template for patterning dense addressable arrays on platforms and / or templates. Is added). Lithography involves dip pen lithography (nanoscale stylus array and stretching DNA), laser-mediated pattern lithography on preformed grooves, and / or unfolded. This can be achieved by using modifications to the magnetic field and other linear field-mediated patterning of DNA.

The ability to perform enzyme-simulating processes requires an accurate understanding of catalyst profiles and the dynamic placement of important chemical and other functional groups in 3D space (WOM Wang et al., "BIOMOLECULAR SIMULATIONS: Recent Developments in Force Fields, reference Simulations of Enzyme Catalysis, Protein-Ligand , Protein-Protein, and Protein-Nucleic Acid Noncovalent Interactions ", Ann. Rev. Biophys. Biomolec. Struc. 30, 211 (2001)). Addressable arrays with regular, geometric patterns significantly reduce the uncertainty inherent in this arrangement, and transient state profiling to optimize the functional action, dynamic conformations of the main catalysts, and catalysts. When modeling, we use less complex finite element analysis and vector-based force field calculations.

Due to this convenience, a catetian (x, y in FIG. 3) or curved (r, pi in FIG. 15) pattern of polynucleobases can be permanently lithographically graphed onto the master. This allows for simple addressing of physicochemical functional groups on the platform, simultaneously and consistently across billions of arrays of this type. As mentioned above, geometrically uniform addresses also simplify computer-based modeling of active site design, orthogonal function behavior, dynamic functional translocation and other activities (W. Warshel and reference: "Insight from Computer Simulations Dynamics of Biochemical and Biophysical Reactions", Quart Rev Biophys 34, 563 (2001)) WW Parson WW 's... Most of these are rendered through finite elements and vector force field based programs that are disambiguated by the irregular coordinates of the spatial coordinates in which the elements and vectors are modeled ( M. Garcia-Viloca et al., "How enzymes work: analysis by modern rate theory and computer simulations", Science 303 (5655) , 186 (2004)).

FIG. 15 shows an example master shape that includes a permanent ssDNA 151 lithographically graphed onto a gold 152.

Guidelines for immobilizing, unfolding, and mounting the ssDNA on the surface

In fact, an important factor in keeping ssDNA unfolded is the agreement between the nature of the unfolded region of the DNA and the artificial modification of the DNA to function as a master substance. As mentioned above, phosphorothioate-backboned ssDNA on gold can be used for permanent templates (A. Csaki et al, "DNA monolayer on gold substrates featuring by nanoparticle labeling and scanning force microscopy". ", Nucleic Acids Res . 29 (16) , 81 (2001). However, strong reducing potential in large amounts of solution may be required to prevent the formation of disulfide bonds with each other before the aS phosphate group unfolds (which may also be required in the chain between nucleobases in the DNA). assists intra-strand and inter-strand hydrogen bonding, and if untwisted, helps to bond between chains and inhibits spreading). During unfolding and loading, the redox potential needs to be shifted to a more oxidized state that promotes covalent bonding of aS and gold. However, this should preferably not take place too quickly, otherwise the DNA can be restrained from prematurely mounting and further unfolding, thus inhibiting the construction of geometrically correct addresses.

Another factor to consider is the formation of a rare surface phenomenon as the ssDNA unfolds and a hydrophobic interface formed by symmetrical potential differences is formed. This is actually a "pseudo-phase" more like a non-coexistent colloid that interacts with a solvated polymer with a true solid state, ie the wall effect. This interface / pseudo-state is formed by the intended backbone that covers the ssDNA and mounts only natural or modified phosphodiester groups on the receptive solid phase. As the ssDNA transitions from the coil wound to the linear polymer, the continuous backbone residue 'points down' and the continuous nucleobases 'point up'. Using each successfully unfolded and mounted residue, the dipole moment of the surface is obtained with higher electron density phosphodiester, phosphorothioate, phosphoramidite group. ) (Or other backbone) and amine-rich, generally increased overall due to the increased total potential difference between the nucleobases of the cations.

More importantly, when ssDNA or RNA is used, a strong hydrophobic ribose group is used to arrange cyclized sugars through ring stacking, water repulsion, and van der Waals interactions. Due to the tendency, they will begin to form "oily mezzanine". This phenomenon occurs on double stranded DNA at the center of the 3D helix and contributes in part to the enormous strength of dsDNA and tensile cohesion to hybridized ssDNA. If the lithography wrapped in high density on the surface, when hybridized with the physiological saline (with a Mg ^{2 +),} mezzanine formed by the complementary strand, for example, directly to the DNA adjacent to the bulk organic phase (bulk Even in the high energy exchange reactions occurring in the organic phase, Watson-Crick bonds will be sufficiently repulsive to be preserved. Although seemingly unusual, this advantageous phenomenon is that various trans-biotic chemical reactions in immiscible solutions (eg, dichoromethane, toluene, or hexane) do not compromise the address on the platform. To be performed.

If PNA is used to lithographic master, a hydrophilic pseudo-state can be formed by stacking nucleobase cyclic groups. However, it does not have uniform stacked deoxyribonucleotides and potential differences are not necessarily formed. If desired, the phenomenon of potential difference may be due to the potential between the nucleophilic tertiary amine backbone of PNA (with any beta-amino acid except proline) and an electrophilic group such as a hydroxy moiety for the solid phase, for example. It can be configured by increasing the difference.

Options for configuring a template from the master

The ssDC template can have the following features to function as a foundation or mold for mass production of catalyst platforms: 1) The template covers the entire 3D negative region (open gaps or spirals) of the final production platform. Assume a positive protruding shape to fill—for each mold unit, a positive protruding shape across the array of templates individually and consistently during production of the platform; 2) a single strand of DNA or PNA on a template is an exact mirror-image pattern of an addressable array on the platform, generating this pattern on the product through complementary base pairing; 3) The template consists of a material that allows permanent integration of DNA, PNA or other nucleobase polymers via backbone covalent bonds that leave nucleobases on the platform free of hybridization and the nucleobases on the platform; And 4) the template does not lose its integrity as a mold that is packed, not bent, or which creates a platform over the expected lifetime. Specifically, the pattern and base chemistry of ssDNA / PNA is preserved on the template over multiple stamping cycles.

The first, third and fourth features do not significantly affect the ability of ssDC to produce catalysts in the near future. The prior art of 3D nanoimprint pattern transfer in a process that proceeds from a hard cast or beam-lithographed originals to a soft cast polymer that hardens into a product, the negative shape of the master It was easy to produce a simulated mold (accurate to less than 1/4 of a cubic nanometer) and easily applied to the technology. Materials capable of surviving thousands of stamp-and-lift or rolling cycles for platform production are also easy to apply to this process and to obtain shapes accurately. Such materials are surface functionalized to allow covalent bonds with artificially modified DNA, resulting in permanently complementary addresses of mirror-images. For example, vapor deposited gold (with titanium on top) is phosphorothioate with sufficient strength to survive thousands of hybridization / dehybridization (ie, inking and stamping) cycles. Permanently bind to the modified DNA. Another option is siloxane functionalized templates capable of permanently binding amine backbone-modified DNA backed by a similarly hard material, such as aluminum, which preserves the integrity of the shape (E. Magni and GA Somorjai, "Electron Irradiation Induced Chemical Vapor Deposition of Titanium Chloride on Gold and on Magnesium Chloride Thin Films.Surface Characterization by AES, XPS, and TPD", J. Phys . Chem . 100 (35) , 14786 (1996) Reference).

However, the second feature represents an important and sophisticated aspect of ssDC, the quality of which will determine whether the platform will undertake catalysts or have a consistent combinatorial ability. In short, the ability to lithographically modify a modified polynucleobase such as ssDNA or PNA with sub-nanoscale geometric precision on the master and with the oligomers presented in the correct axial orientation for hybridization is a template This is a clue to enabling the technology to construct.

The best understood, available and applicable options for current template construction are: A) dip pen nanolithography (DPN) of ssDNA for 3D shapes; B) femtosecond laser-tuned patterning of ssDNA through a sacrificial layer; C) laser-tuned lithography for preformed grooves on the 3D surface; And D) magnetically-mediated polynucleobase arrangement across the surface.

Option A. Master Configuration by Direct Lithography

One direct way to place ssDNA on 2D or 3D planes is to use deep pen nanolithography (JH Wei and DS Ginger's "A Direct-Write Single-Step Positive Etch Resist for Nanolithography", Small 3 (12) , 2034 (2007); and D. Bullen et al., "Parallel dip-pen nanolithography with arrays of individually addressable cantilevers", Appl . Phys . Lett . 84 (5) , 789 (2004). An atomic force microscope (AFM) stylus is used as a partially adsorptive electrostatic solid phase that attracts charged polymer. ssDNA is modified at the end for end-specific anchoring to the determined starting point (which actually preserves the identification of the DNA strand by determining its length), and then It can unfold into it. Many ligands have end-functionalization of ssDNA, including biotin (for high affinity binding to streptavidin) and digoxigenin (for liganture to antibodies) (end-functionalization) can be modified.

After fixation, the ssDNA can be pulled across the 2D plane based on proper management of the following physicochemical factors: 1) induced and intrinsic charge of the AFM tip; 2) the inherent charge of the polymer, a function of charge density, counter ions, and polymer dipole moment. The polymer dipole moment depends on the orientation of the polymer relative to the AFM tip, especially when the moment vector is orthogonal to the long axis, as in the case of ssDNA; And 3) the electrostatic and frictional nature of the surface, including solvation, charge, charge density, and non-uniform potential caused by a "completely uneven" surface (hence the ssDNA loading energy).

These factors contribute or inhibit the transition from the coiled coil of the ssDNA strand to a linear form approaching its theoretical maximum length. When applied to heterogeneous 3D surfaces, other factors act as described below. However, when using DPN on homogeneous 3D surfaces such as the master shape as described above, general considerations of polymer dynamics and material quality are generally sufficient to describe the key factors. This includes the following: (i) unfolding ssDNA by force sufficient to unfold but not destroying ssDNA or by coupling between the ssDNA and the anchor at the end; (ii) spreading the ssDNA in the (+ z) vector component, ie “upwards”, especially over the 3D elevation; (iii) a change in the potential vector and its magnitude and direction, which are contoured in particular, in the transition between the flat point and the curved point where the potential tends to fit; And (iv) potential repulsion or attraction caused by the presence and proximity of ssDNA already lithographed when lithographically at high strand density.

FIG. 16 shows ssDNA 151 recursively fixed on receptor point 163 spotted with DPN and linearly unfolded over template shape 152 to produce a Cartesian pattern.

Option B. Femtosecond Laser Fixation Under the Sacrificial Layer

An alternative to the fixed function DPN-mediated spotting is to use a femtosecond (Fsec) laser to remove the sacrificial layer region of the material acting the 2D surface and / or 3D contour intended as the master mold. (Femtosecond pulsed laser micromachining of single crystalline 3C-SiC structures based on a laser-induced defect-activation process, Y. Dong et al . , J. Micromech . Microeng. 13 , 680 (2003); G. Pellegrini et al. "Technology development of 3D detectors for high -energy physics and imaging", Nucl. Instr. Methods Phys . Res. Section A: Accelerators , Spectrometers , Detectors and Associated Equuipment 487 (1-2) , 19 (2002); And "Direct Fabrication of Micropatterns and Three-Dimensional Structures using Nanoreplication-Printing (nRP) Process", SH Park et al., Sensors and Mater . 17 (2), 65 (2005)). The advantages of this approach over DPN include more accurate, faster and eksetns placement of smaller anchor points, which reduces the likelihood of more than one strand bond occurring in a given location, and the sacrificial layer is a mask-based lithography material. It has more diverse components than (located under the sacrificial layer and forms a 3D shape), allowing better control over surface energy, solvation and friction factor. Overall, they can improve the ability to unfold and mount ssDNA.

A complementary ssDNA, 25 billion strands of 100-mer (mer) with a given 20 nm spacing density could potentially be lithographically graphed onto a master of 1 square centimeter (500,000 on one side). Although there is no estimate of the speed of the DPN technique for generating these many points, in theory the laser of Fsec generates many such anchor points on the sacrificial layer in about an hour.

Fsec lasers are a relatively common technique and are widely used in the microchip and nanolithography industries. Spatial control is accurate to ± 0.1 nm and energy deposition (exposure time) is accurate to ± 0.1 fsec (100 attoseconds). Targeting is generally controlled in place by a computer-controlled potential difference on a diode-based rasing element that yields a quantum effect of changing the emission vector with extreme accuracy. This does not require mechanical rearrangement of the laser (a factor that cannot be accurate to ± 1 nm) and as a result no moving parts are provided. Also, no liquid flow based structure is required. All these disadvantages are factors in DPN-adjusted spotting of the anchor point. Overall, the use of Fsec lasers makes it possible to remove hundreds of millions of victimized points accurately and evenly in a very short time.

FIG. 17 illustrates the unfolding of the ssDNA similar to the scheme shown in FIG. 16. ssDNA 151 is repeatedly fixed on laser-depleted point 173 of Fsec by functionalizing with SA or anti-DIG 174 on sacrificial layer 175. And spread linearly across the master shape 172. Contrasted is a method of fixing the end of the ssDNA (DNA of FIG. 16, Fsec laser of FIG. 17) and the surface to be mounted. Appropriate master shapes can be located underneath the sacrificial layer to form a 3D ellipsoidal hemisphere, which acts as a receptor for common ssDNA end functional groups such as streptavidin and anti-digoxigenin antibodies. Can be modified to

Option C. Configuration of the master by laser on grooved surface

Whatever method of immobilizing ssDNA is used, the additional factors inherent in the sacrificial or lithographic layer are axes that can improve straightening (for Cartesian addresses) or curved (for curved addresses) patterning and unfolding and alignment. Options for recesses or grooves in the direction. In short, the linear structure of a charged or stretch-resistant polymer, such as ssDNA, is suppressed if the other structure is suppressed by a factor appropriately described as "wall effects". More plausible ("Conformational Analysis of Single DNA Molecules Entropically Induced Motion in Nanochannels" by JT Mannion et al., Biophys , J. 90 , 4538 (2006); and "Gradient nanostructures for interfacing microfluidics and nanofluidics", Appl. . Phys. Lett. 81 (16 ), 3058 (2002)). For example, functionalizing the walls with hydrophobic groups (enough to repel charged nucleobases, but not enough to attract deoxyribose groups) may further maintain the linearized polymer structure. (HS Choi et al., "Photopatterning of gold and copper surfaces by using self-assembled monolayers", Curr . Appl . Phys. 7 , 522 (2007); and "UV Photopatterning of Alkanethiolate Monolayers Self-Assembled on Gold" by MJ Tarlov. and Silver ", J. Am . Chem . Soc. 115 , 5305 (1993)). The "floor" of the groove can be suitably functionalized to the type (s) of the backbone on the polymer, for example a gun for electrostatic bonding of phosphodiester backboned ssDNA with ordinary anions. Cationic charge; Gold for {aS-to-Au binding} for phosphorothioate-modified ssDNA; Or appropriately functionalized with coulomb bufered siloxane for amine-modified ssDNA.

The grooves make an important contribution so that more complex patterns can be produced by light- or electron-beam lithography. This concept may introduce Fsec laser-based spotting of anchor points for repetitive addition of uniformly end-functionalized DNA in the strategy of “sacrificing, functionalizing, adding DNA, repeating ... unfolding”. For example, curved grooves can be cut into each of billions of master units in the array. 18 shows an ssDNA 151 that is repeatedly anchored on an Fsec laser-depleted point 173 on a 2D surface and a 3D master contour 184 and curved or linearly unfolded in a pre-lithographic groove 185. To show. The anchor points of strands 4 to 11 describe lithographic confinement grooves that implement a conformational change of the ssDNA in an elongated form in the coil wound prior to being placed over the 3D mold contour. This figure suggests an unfolding method of ssDNA based on translocating the other end of the polymer through a magnetic or weighted polymer bead across the radius inside the groove, which is a DPN Independent of unfolding through This will be described in detail in Option D below.

Importantly, the surface may be patterned with extensions or protrusions rather than grooves. Current technology mask-based lithography introduces, for example, the deposition of gold linearly or in other patterns on titanium, or vice versa, except for the required catetian or curved pattern for the mounting of phosphorothioate ssDNA. Electromagnetic energy can be used to remove gold.

Spreading the ssDNA from the functionalized anchor to the distal point can be accomplished by paramagnetic beads of about 25 nm in diameter or latex beads of the same size. Electromagnetic and / or optical fields (which are through laser “tweezers”) can be used to move the beads relative to the grooved or raised pattern. The electro-optical properties of field arrays that can achieve this spread are described in option D.

Option D. Electromagnetic Arrangement on Contoured Surfaces

Finally, whatever method for anchoring ssDNA is used, and regardless of the nature (or absence) of the sacrificial layer and its contours, the method of spreading ssDNA across or on the 3D surface, This can be important in the configuration of the master.

Considering the lithographically ssDNA, two methods of unfolding the polymer are used: As shown in FIG. 18, a continuously confined tunnel proceeding to a recess formed with a narrow groove sculpted into a 3D shape. linear field pulling the ssDNA strand anchored into the confining tunnel; And / or varying vector fields pulling the beads around and inside the grooves. For the latter, the magnetic or optical field can be rotated around a static master, or vice versa, on which the master can be fixed on a table that is associated with a linear electro-optical field.

Appropriate magnetic fields can be generated by permanent magnets where the geometry of the field is limited to linear distances, or by electromagnets that can be programmed to produce linear uniform fields. The electromagnets can be dynamic, enabling translocation of sensitive beads to pull ssDNA.

19 is a confined tunnel from the Fsec laser-lithographic anchor point 173 to a groove or protrusion 185 25 to 40 nm wide, with one ssDNA strand through a linear vector magnetic field line 196. Shows the unfolding. Removing the sacrificial layer with multiple Fsec laser pulses may then reveal the underlying layer, which may be functionalized with digoxigenin, for example. In this example, anchoring only one DNA strand can be accomplished by addressing a prefabricated structure comprising components of the following (1: 1: 1) molar content ratio (5 'to 3'). Orientation): latex beads 197 coated with 50 nm anti-digoxigenin; 5'-DIG --- {ssDNA sequence}-3'-biotin 191; And beads 198 coated with 25 nm superparamagnetic streptaavitin.

Again, the grooves can be replaced by protrusions, so that there is no need to change the way of spreading. However, if the advantages of the restriction wall to help keep the ssDNA in a linearized structure are not provided, it is wise to perform the dilation protocol more diligently. This strategy actually creates a master that facilitates platform creation because it is easier to stamp positive shapes into soft cast polymers than by stamping negative shapes into polymers that are expected to flow even if not guaranteed. . The latter method is similar to the Intaglio printing process (with obvious uncertainty inherent when performed in nanoscale).

An option to reduce the deformation of the DNA backbone from forming covalent bonds to the solid phase before certain residues are unfolded is to use other temporary enclosures that "spool" the charged polymer to prevent premature surface adhesion. enclosures or ssDNAs that are not wound in liposomes. Since the relatively "smooth" colloidal enclosure with the intended surface properties is easier to move relative to the contour than the potentially "sticky" wound mass of polymer, this method implements translocation for limited space. Has the added advantage. However, since longer strands require larger liposomes or other enclosures, the enclosure is limited to fairly short ssDNA segments (250-mers or less, but this is sufficient for most small active site simulations). Has This will potentially block the groove. Certainly, it does not matter if the spreading is performed in a lithographically projected beam unless the fatty acid or other colloidal content of the enclosure reacts with the rising surface or is separated from the enclosure. Gold is not particularly contaminated with fatty acids of anions or cations, and is a surface whose enclosure does not react with gold or an intervening surface, ie a surface composed of a structure such as a micelle covered by an immiscible scaffold. The problem is partially improved when it is provided.

Colloids having strong, induced dipole moments, and superparamagnetic and slightly ferromagnetic (ferromagnetic are polymers mostly functionalized with magnetic beads) are usable and can be used to form uniform vector magnetic moments. After cross-linking or polymerization in heterogeneous mixing of different colloids, exposure to a linear magnetic field of about 1 Tesla is caused by another magnetic field used to align and unfold the magnetic moment of the monomo. Will create a structure that can be transposed. Alternatively, the enclosure can be decorated or attached to superparamagnetic beads for simpler manipulation, translocation, and unfolding of the DNA.

Besides the arrangement of the magnetic moments, there is another important need for the partial polymerization of "magnetosomes". As ssDNA is pulled out of the enclosure by unfolding and mounting, dissolved molecules are required to enter inward from the bulk phase in order to equalize the hydrostatic pressure on the enclosure. Fatty acids and other long chain molecules that form the nearest neighbor covalent bonds when subjected to UV light have a porosity that allows the DNA to escape and water molecules and salts to enter. It can be used to form a spherical lattice such as As a result, a sphere of enclosed ssDNA in a decreasing aqueous environment, ideally protected by a hydrophobic core adjacent to the non-reactive aqueous periphery, is ideally obtained, the entire complex being inside the groove or lithography It may be transposed by a induced magnetic field to be pulled over the bulge.

20 illustrates the unfolding of phosphorothioate modified ssDNA strand 201 into lithographic gold 202 (with outline 203) by unwinding from an enclosure of colloids that can be translocated in magnetic field 196. Illustrated. The beads 207 may comprise liposomes made of a hydrophobic core 204 and a hydrophilic periphery 205 scaffolded with a lattice superparamagnetic polymer doped with a lipid mixture. The unidirectional magnetic moment of the scaffold (Cateshian pattern) can be induced by a field of about 1 Tesla or greater after polymerization in UV light to form a hollow shell. Alternatively, larger (eg, 250 nm diameter) paramagnetic beads can be functionalized with the same liposome enclosure. In this case, the lattice work may function to mechanically hold the enclosure.

The present invention has been described as a single strand dimensional construction method. The above detailed description is merely for explaining the application of the principles of the present invention, the scope of the present invention will be determined by the claims in consideration of the detailed description of the invention. Other variations and modifications of the invention will be apparent to those skilled in the art.

Claims (26)

In the process of generating a three-dimensional addressable array of biopolymer nucleic acid,
a. Providing a nanoscale three-dimensional master shape having a surface; And
b. Coupling a plurality of master biopolymer nucleic acids to a surface of the master shape in a regular pattern. The method of claim 1,
The binding step b) comprises the step of binding the master biopolymer nucleic acid to the surface of the master shape using dip pen lithography (3D addressable) Process of creating an array. The method of claim 1,
The step of binding b) comprises binding the master biopolymer nucleic acid to the surface of the master shape using laser-mediated pattern lithography. Process of creating a dressable array. The method of claim 3, wherein
Wherein said surface comprises a plurality of grooves or protrusions, and each of said master biopolymer nucleic acids is coupled to said grooves or protrusions. The method of claim 1,
A susceptible mass is attached to the ends of each master biopolymer nucleic acid, and the binding step b) comprises binding each master biopolymer nucleic acid to the surface of the master shape using magnetic field patterning. A process for generating a three-dimensional addressable array of biopolymer nucleic acid, characterized in that. The method of claim 1,
Wherein said nanoscale three-dimensional master shape comprises a cylinder, a trough, a cleft, or a compound helix. The method of claim 1,
The master biopolymer nucleic acid is a step of generating a three-dimensional addressable array of the biopolymer nucleic acid, characterized in that it comprises DNA, PNA, RNA or LNA. The method of claim 1,
Hybridizing a plurality of complementary template biopolymer nucleic acids to a plurality of master biopolymer nucleic acids on the master shape. The method of claim 8,
Binding a surface of the template shape to the plurality of complementary template biopolymer nucleic acids and removing the template shape from the master shape with the associated complementary template biopolymer nucleic acid. A process for generating a three dimensional addressable array of nucleic acids. The method of claim 9,
Template bonding comprises impression molding comprising a three-dimensional addressable array of biopolymer nucleic acids. The method of claim 10,
A process for generating a three-dimensional addressable array of biopolymer nucleic acids, characterized in that the template array to be simulated is delivered by roller or flat stamp. The method of claim 9,
Template bonding comprises pressure molding. The process of creating a three-dimensional addressable array of biopolymer nucleic acids. The method of claim 12,
The press forming comprises the step of applying a pressure to an elastic material consisting of a bladder (bladder). The method of claim 9,
Hybridizing a plurality of complementary platform biopolymer nucleic acids to a plurality of template biopolymer nucleic acids on the template shape, wherein the three-dimensional addressable array of the biopolymer nucleic acid is generated. The method of claim 9,
Binding a surface of the platform shape to the plurality of complementary platform biopolymer nucleic acids and removing the platform shape from the template shape with the associated complementary platform biopolymer nucleic acid. A process for generating a three dimensional addressable array of nucleic acids. The method according to claim 1 or 15,
Hybridizing the master or platform biopolymer nucleic acid with a complementary probe comprising reactive chemical groups. 17. The method of claim 16,
Wherein said reactive chemical group comprises an amino acid, wherein said three-dimensional addressable array of biopolymer nucleic acids is generated. 17. The method of claim 16,
Wherein said reactive chemical group mimics an enzyme active site. 17. The method of claim 16,
Wherein said reactive chemical group provides a catalyst, wherein said three-dimensional addressable array of biopolymer nucleic acids is provided. A three-dimensional (3D) addressable array comprising a plurality of biopolymer nucleic acids bound in a regular pattern on a surface of a 3D shape. The method of claim 20,
And the surface comprises a metal, a plastic or a polymer. The method of claim 20,
3D addressable array, wherein the metal comprises gold. The method of claim 20,
And a complementary probe hybridized to each of the plurality of biopolymer nucleic acids, each complementary probe comprising a reactive chemical group. 24. The method of claim 23,
3D addressable array, characterized in that the reactive chemical group is an amino acid. 24. The method of claim 23,
The reactive chemical group is a 3D addressable array, characterized in that to simulate the enzyme active site. 24. The method of claim 23,
Wherein said reactive chemical group provides a catalyst.

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