CA2406948A1 - Biochips, preparation and uses - Google Patents

Abstract

The invention concerns biochips, their preparation and their uses. In particular it concerns biochips comprising immobilised nucleic acids on a support by means of a dendritic arm and/or an arm bearing a negative charge and/or directly on supports bearing a negative charge. The inventive methods and biochips can be used for detecting or analysing gene expression, for searching genes of interest, or for diagnostic purposes, for example.

Description

BIOCUCES, PREPARATION AND USES
The present invention relates to the field of biology and genetics.
It relates in particular to new compositions and methods for the preparation of biochips and their uses. It concerns in particular biochips comprising specific nucleic acid populations and / or prepared from molecules of anchors or specific supports. The methods and biochips according to the present invention can be used to the detection or analysis of gene expression, for gene search of interest, or for diagnostic applications, for example.
DNA microarrays or, more generally, your microarrays nucleic acids ("biochips") are miniaturized systems of analysis large-scale genetics, for example to study activity transcription of a large number of genes (expression analysis gene), to determine the sequence of a large number of DNA fragments (including genetic polymorphism analyzes), etc. Their general principle consists in:
- Fix in an orderly manner fragments of nucleic acids on a support, and this in a miniaturized way in order to be able to fix a large number of fragments different on a reduced surface. The biochip is the set made up of the support and the nucleic acid fragments attached to this support. In the present invention, the nucleic acids attached to the biochip are referred to as name of "targets". Each corresponding nucleic acid (or group of nucleic acids) at a specific DNA or RNA sequence is attached to a specific location in the support ("Spot"). Preferably, the biochips have a density greater than 500 spots per cm2, preferably more than 1000 spots per cm2 and even more preferably greater than 5000 spots per cm2.
- Hybridize on the chip a population of nucleic acids to be analyzed (which whatever their nature and their biological origin). Here we designate the acids nucleic acids to be analyzed under the name of "probes". During hybridization, the acids nucleic acid present in the probe will specifically bind to targets present on the chip, whose nucleic sequence is similar to all or part of the sequence of these probes,

2 - Measure the quantity of specifically hybridized probes on each of the chip targets. This measurement can be carried out either by marking fluorescent or radioactive prior to the probes and reading of the quantity of labeling present after hybridization on each target, either by other types of measurement of the amount probe-target hybridization for each target (such as, for example and way non-exhaustive, the measurement of micro-currents induced by the formation of a capacity electric double-stranded target probe, or direct mass measurement molecular of probe attached to each target).
A number of biochips have been described. in the prior art.
However, these biochips have a number of disadvantages or limitations related in particular to the nature of the target nucleic acids used and or the preparation conditions for biochips, which make their production and their difficult to use.
So the single stranded nucleic acid chip technology developed by the company Affymetrix consists in synthesizing oligonucleotides directly on chip support, by photolithography and DNA synthesis in phase solid. This principle, which can be obtained with other synthetic methods, is commonly referred to as "in situ synthesis". However, at this day, this principle does not make it possible to synthesize with sufficient efficiency oligonucleotides more than 25 bases long, and the use of such oligonucleotides (25 bases or less long) leads to the presence of numerous hybridizations nonspecific probes, and therefore poor reproducibility of experiments using this system.
In this regard, it is known that the specificity of hybridization between two nucleic acid fragments (e.g. the probe-target complex for biochips) depends on the conditions under which the hybridization reaction is done, the base composition of these nucleic acids, their similarity or identity identical sequence length. So the longer the length of identical sequence between the two fragments, the greater the hybridization specific. However, beyond a certain length, variable depending on of WO 01/4450

3 CA 02406948 2002-06-14 pCT ~ R00 / 03572 sequences, but in general on the order of more than 1000 bases, the structures secondary molecules can interfere with reaction yield hybridization (folding of the molecules on themselves, even hybridization of the molecules on themselves).
It would therefore be useful to have technologies enabling the production of biochips comprising any type of nucleic acids, under conditions compatible with an efficient and sensitive reading of hybridizations. However, the fixation of nucleic acids on biochip supports remains problematic to date, mainly due to steric hindrance problems and current physico-chemical methods of fixing to supports.
The question of steric hindrance linked to the deposit of a large number of molecules on a very small surface ("spot" of each deposit on the chip) is important. It is indeed necessary that the deposited molecules (targets) are in number and in sufficient density to allow the measurement of hybridization thereon of nucleic acid molecules to be analyzed (probes). However, do not than this density is such that the access of the probe molecules to the target molecules is hampered by the spatial obstruction of the targets.
Furthermore, the conditions for fixing the targets on the biochip are also important. Indeed, this fixation must be stable, ideally from covalent type, without however reducing the capacity of the targets to be hybridized sure respondents. From this point of view, ideally, setting targets is performed by one of the ends of the target (5 'or 3') and not on their length.
Finally, for questions of both steric hindrance and availability of targets along their entire length, but also of technical feasibility, the question is not resolved whether it is better to fix the targets directly on the support of the chip or if it is better to fix them there by means of an "arm" (molecule whatever its nature and size).
The present invention now provides advantageous solutions to difficulties and limitations of the techniques and products of the prior art.
These solutions relate in particular to the conditions for fixing targets on the support,

4 as well as on the nature and / or the physico-chemical characteristics of molecules used. The invention also describes biochips on which of particular populations of target molecules are deposited, in particular target nucleic acid populations of defined size.
The present invention describes in particular a new fixing approach of target nucleic acids on biochip carriers. It can be put in works with any type of target nucleic acids as defined below, and with any type of support, as defined below. It can also be used in works equally well with target nucleic acids synthesized directly on the biochip support (in situ synthesis) or synthesized independently then fixed on the biochip in a second step. In addition, it can also be put in works with molecules of interest other than polynucleotides.
More particularly, according to a first aspect, the present invention resides in use, to fix the targets on the chip, arms (or molecules "Spacer" or "linker") having a spatial structure (primary structure or secondary of the molecule) in the form of a tree. In particular, the invention generally concerns the use of any molecule whose structure is organized according to a tree structure principle, whether this is one or several degrees of freedom, as "arms" for fixing targets on a support of biochip.
Thus, a first object of the present invention resides in a biochip, characterized in that it comprises nucleic acids immobilized on a support by means of tree-shaped arms.
Another aspect of the present invention resides in a compound biochip of single-stranded nucleic acids attached to a support, in which the acids single-stranded nucleic acids are between 25 and 100 nucleotides in length approximately, preferably between 30 and 60 nucleotides approximately.

Another aspect of the invention also resides in a biochip comprising nucleic acids immobilized on a support carrying a charge uniform negative electric or by means of arms carrying an electric charge negative.

5 The invention also relates to the preparation and use of biochips as defined above, for genetic analyzes, sequencing, gene research, diagnosis, etc.
To allow a better understanding of the present invention, the The following definitions are provided. Unless otherwise specified, other terms techniques used in this application must be interpreted according to their usual sense.
Biochip: A biochip is understood in the sense of the invention of any support on which are deposited target nucleic acids. Generally, acids nucleic acids are immobilized on the support, preferably by bonding covalent. They can be immobilized directly on the support, or indirectly, through through "arm". They may be biochips for which the targets have been previously produced and then fixed on the support, or for which the targets are synthesized directly on the support (including by photolithography and solid phase DNA synthesis). The biochips according to the invention can understand a low or very high number of targets, and the size of the total surface of the support occupied by the targets can be variable.
Generally, the term "arm" (or "spacer" or "linker") denotes any molecule which can be used to attach target nucleic acids to a support. These are molecules incapable of specifically forming of hybrids with probe nucleic acids. It is therefore preferentially of molecules of essentially non-nucleic nature. In the constitution of biochips of the invention, a single type of “arm” or even “spacer” or "Linker" can be used by biochip or, mixtures of structural arms different. However, it is generally preferred to use a single type of arm sure WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

6 a single biochip, so as to obtain a homogeneous signal. For exemple, the arm molecule can be an organic polymer of protein nature, carbohydrate or lipid, or a synthetic inorganic polymer.
In the context of the present invention, the "target" nucleic acids present on the biochip support can be of nature and origin various.
Thus, it can be single or double-stranded nucleic acids, of origin natural, synthetic and / or semi-synthetic. In particular, it may be oligonucleotides synthesis, PCR products, genes or gene fragments, plasmids, single or double-stranded cDNA, RNA, etc. It can be populations acids nucleic acids isolated from biological samples, such as biopsies, of samples of mammalian, especially human, cells, tissues or organs, samples of plant, animal, viral or bacterial origin, etc.
Regarding of oligonucleotides, they are more particularly defined as fragments single stranded nucleic acids between 25 and 100 nucleotides, whether these are DNA or RNA in nature.
The biochip support can be varied. So it can be any support adapted to receive, directly or indirectly, acids target nucleic acids. In particular, the support may include a flat surface and or domed, and be solid or semi-solid in nature. In addition, the support can to have variable shape and size. Mention may in particular be made of the supports circular, rectangular, square, etc. The surface of the support is preferentially between 300 and 3000 mm2, preferably between 400 and 1800 mm2. As example, we can cite as material entering into the constitution of supports glass, silica (especially silica in glass after deprotection by ammonia), poly-lysine, amino-silanes and / or amino-reactive silanes.
The appended figures illustrate embodiments of the invention and more specifically describe Figure N ° 1: Use of tree target fixation arms;
The use of non-arborescent target fixation arms WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

7 Figure N ° 2: Uridine diphosphate glucose (UDPGIuc), according to Harper biochemistry, RK
Murray, DK Granner, PA Mayes, VW Rodwell, Publisher Mc Graw-Hill International (UK) Ltd ..
Figure N ° 3: N bond within a glycoprotein.
Figure N ° 4: O bond within a glycoprotein.
Figure N ° 5: Anchoring of the capture probes on the biochip by the intermediary of glycogen molecules (scale not respected).
Figure N ° 6: Anchoring of the capture probes on the biochip by the intermediary of glycogen molecules (scale not respected).
Figure N ° 7: Structure of the glycogen molecule at a point of branching out, according to Harper Biochemistry.
Figure 8: Amylopectin showing branching 1 - ~ 6, according to Harper Biochemistry.
Figure N ° 9: Schematic representation of cartilage agrecan bovine nasal, according to Harper Biochemistry.
Figure N ° 10: Schematic representation of polymers immunoglobulins human. The polypeptide chains are represented by lines thick;
the disulfide bridges connecting the different polypeptide chains are represented by fine lines.
As indicated above, a first object of the present invention resides in a biochip, characterized in that it comprises nucleic acids immobilized on a support by means of tree-shaped arms.

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8 This principle, new to our knowledge, has the advantage of being able limit the difficulties linked to the steric hindrance of the molecules. In indeed the fact to fix the target nucleic acids at the end of the tree of such "arms"
increases the presentation surface of these acid targets nucleic probes studied (see Figure 1). It also reduces the number of molecules to be fixed directly on the support of DNA chips (only the trunks of these "arms" are fixed). This therefore makes it possible to obtain a high density of acids.
target nucleic acids on each "spot" of the chip while having a space requirement steric of these targets decreased compared to situations where targets are is directly fixed on the support, or fixed on the support by through a linear "arm".
Several physical natures of arms have been proposed in the prior art for fixing oligonucleotides on supports. We can cite, in a way no limiting, linear oligoethylene glycols from 26 to 105 atoms of length, selected for their ability to be covalently linked to one of ends of the oligonucleotide on the one hand, and on the other hand to be fixed so covalent to various biochip supports. However, to date, all of the arms that have been proposed have a linear primary structure and / or are of a synthetic nature (see WO
00/43539 and WO 99/10362). The present invention lies in the implementation evidence that nonlinear arms can be used to fix acids nucleic acid supports, in particular tree-shaped arms, preferably based of a polymer of biological origin.
In a more particular embodiment, the arm is a polymer having a spatial organization in tree structure, preferably oblong.
Advantageously, the arm is a branched polymer, having one or more levels branching.
The tree arm can be prepared from different molecules organic, in particular organic polymers (existing in nature), their derivatives, or mixed compounds, comprising an organic part and a part synthetic.

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9 In a particular embodiment, the arm according to the invention includes an organic polymer of biological origin. The biological compound used for the preparation of the arm can be a sugar, a polypeptide, a glycoprotein, glycopolypeptide, immunoglobulin, etc. eventually under isolated, complexed or multimerized form, if necessary functionalized or amended, in particular by means of synthetic molecules or polymers, or of groups reactive chemicals.
As organic compounds existing in nature, these can be purified from organic or synthesized extracts artificially.
According to a first advantageous embodiment, the polymer organic is a polymer of sugars or polysaccharides.
The use of such sugar polymers for the preparation of biochips or, more usually immobilization of nucleic acids has multiple interests and advantages, linked in particular to their chemical properties and their structure primary and secondary.
So polysaccharides are hydrophilic, which is a condition favorable to the hybridization of the probes on the targets and in particular that of the probes of nature nucleotide (DNA, RNA) or derivative (PNA). Indeed, these are hydrophilic and their hybridization is done in aqueous solution. A polymer anchor hydrophobic would therefore risk hindering hybridization by having a repellent effect on the hybridization solution.
On the other hand, polysaccharides, in particular those described, have radicals allowing the creation of covalent bonds at the chain ends (at all ends), due to the presence of hydroxyl functions (OH). Connections covalent, can be, for example, phosphate bonds (CPC or CPP-VS
or C-Pn-C) as it exists in nucleotide sugars or in sugar donor polynucleotides involved in the synthesis of polysaccharides and glycoproteins. A possible example of anchoring is based on the stages of natural glycogen synthesis and in particular the reaction of glucose-1-phosphate with uridine triphosphate (UTP) in the presence of the enzyme UDPGIuc-pyrophosphorylase to form uridine diphosphate glucose (UDPGIuc, WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572 Figure 2). It can also be nitrogen bonds (CNCC) such that they exist in glycoproteins (see Figure 3), oxygen bonds (COC) How is it also the case in certain glycoproteins or in the structure of polysaccharides (see Figure 4). This property therefore allows to establish so 5 simple covalent bonds between polysaccharides and other polymers (such that, for example, proteins, other polysaccharides, or acids nucleic acids).
The formation in nature of "nucleotide sugars" complexes, by example

10 during one of the intermediate stages of the natural synthesis of glycogen as a glucose donor (in this case, the sugar-nucleotide bond is a diphosphate bond, resulting in uridine diphosphate glucose, with a bond glucose-ribose, see Figure 2) illustrates this type of interaction and the establishment of covalent bonds between sugars and nucleic acids. As for example, the polysaccharide-polynucleotide link can involve type links 1 - ~ 5, sugar1-sugar2, sugar 1 being at the end of the polysaccharide, sugar 2 being that of the nucleotide located at the 5 ′ end of the polynucleotide.
Another advantage of using polysaccharides according to the invention derived from their primary and secondary structure. Indeed, the polysaccharides used in the present invention are polyramified (see Figure No. 5). Otherwise, their size (21 nm diameter for glycogen) is perfectly suited to the miniaturized format of DNA chips, and the gap between branch lines (about 13 residues of glucose natural glycogen) helps to avoid advantageous, too much steric hindrance of the targets fixed on the ends of branched polymer. A branch every 2 to 5 residues, would risk, in effect of interfere with the hybridization of the probes. On the other hand, polysaccharides, such as glycogen, have a spherical shape adapted to the surfaces, according to the invention, of presentation of the probes (see Figures 5 and 6).
Finally, the use of polymers based on sugars also makes it possible to fix, of covalently and through one of the chemical bonds as described previously, the sugars supporting the chip, either by any of WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

11 their ends, or, preferably, by their core. This link can be a direct bond on the support or an indirect bond via of a anchor molecule, such a molecule possibly being, in particular, of a nature protein. So, in the case of natural glycogen, it is linked in a way covalent by its nucleus to a protein, glycogenin (in this case here by a tyrosine residue). In this case, the polysaccharide is therefore used in these applications in the form of a glycoprotein.
A specific example of such a molecule is the glycogen molecule, including endogenous glycogen in humans. This molecule is a polymer of sugars including, from a common core, many ramifications extend, themselves polymers of sugars. A particular object of the present request therefore relates to the use of glycogen or glycogen derivatives in this goal.
Glycogen has all of the properties listed above. She is available commercially and inexpensively (See Figures 5, 6 and 7: chip-glycogen-polynucleotide). A glycogen molecule can have more than 60 ends free carbohydrates, allowing to fix more than 60 probes, which correspond to a very high fixing capacity, and very difficult to obtain by synthesis artificial polymers, while keeping a limited space requirement which is compatible with the intended use according to the invention.
Other examples of such compounds based on organic sugars are amylopectin or any other polymer derived from the structure of starch (see Figure n ° 8), glycosaminoglycans (in particular mucopolysaccharides), as well as any other polysaccharide, whatever the sugar or sugars entering its composition (galactose, glucose, mannose, fucose, xylose, Acetylgalactosamine, N-acetylglucosamine, etc.), which may have a branched structure.
The tree organic polymer or compound can also be a glycoprotein-based polymer. Such molecules exist in nature and have the property of having a structure presenting a protein nucleus, around which can be covalently linked to polysaccharides (O- or NOT-depending on the amino acids which serve as residue and on which the chain WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

12 polysaccharide is fixed) (see Figures 3 and 4). These polysaccharides can be fixed in variable number, and in particular in number greater than 2. The same glycoprotein can have several polysaccharides differing in length, in number of residues between ramifications and chemical residues (pentoses, hexoses etc.) without this affecting the general principle of their use in these applications. The general structure of these molecules can to be branched. In this case, the capture probes (DNA, RNA, PNA, etc.) are covalently attached to the end of the polysaccharide chains. The bond glycoprotein on the chip carrier can be done either at the core protein, or at the level of a polysaccharide chain, but, in this case, preferably at the level of the protein nucleus, and even more so favorite by an O- or N- bond with an amino acid residue, either to a molecule anchoring intermediary itself directly fixed on the support, or directly to the support.
These molecules have the properties required for their application in the framework of the biochips according to the invention, as is the case of the glycogen complex-glycogenin previously mentioned.
In the case of other glycoproteins, the spatial characteristics may vary (size of the molecule, number of branches, and number of residues between the ramifications), but they remain in the same orders of magnitude (factor 10) than those cited for glycogen or agregane, and are therefore compatible with their use for biochips.
The present invention also works with glycoproteins having several polysaccharide chains, in the case where these chains do not are not themselves branched, but where the external structure of the molecule remains globally spherical and has numerous attachment sites at the periphery of probes (general “sea urchin” type structure), and a molecular size of the even order of magnitude as that of glycogen or aggregate.
Particular examples of such compounds are proteoglycans, proteins containing covalently linked glycosaminoglycans. In these VVE 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

13 molecules, proteins form the “protein nucleus” of the molecule, and glycosaminoglycans form its ramifications. Preferably, the invention relates to the use of aggrecan. Aggrecan (see Figure 9), is a glycoprotein naturally present in particular in the cartilages. She can appear in the natural form of multiple proteoglycan molecules aggregated on a central motif (polymer), such as hyaluronic acid, this aggregation can be done, via covalent bonds or no, with the central polymer, and resulting in the formation of a "super molecule"
multi-branched, whose general structure properties are compatible with the invention, and whose free ends, which are sugars, can be related to targets. Finally, proteoglycans are generally negatively charged, this which can promote the specificity of probe-target hybridization in the case where these are nucleic acids (DNA, RNA).
In another particular embodiment, the polymer can also be a polymer of amino acids, or of any type of molecules having amine groups reactive to nucleophilic attack or other types of chemical groups which can be covalently linked to an acid nucleic acid.
The organic polymer can thus be a polymer based on proteins or protein complexes (formed of several subunits, themselves linked Between them by covalent bonds such as disulfide bonds), which present a branched type structure.
A particular example of a polypeptide compound is represented by the immunoglobulins (1g), whether these are in simple form or in the form of complex (as in the case of type A immunoglobulins, see Figure n ° 10).
Immunoglobulins, natural molecules, are easy to produce in large quantities quantity after selection of suitable clones (monoclonal immunoglobulins).
In this application, the immunoglobulins are used as molecules anchoring the capture probes. The probes are linked to the Fab ends of immunoglobulins, either by a classic antigen-antibody bond, in the case where the immunoglobulin is directed against a portion of the probe, that is, of WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

14 preferably by a covalent bond of amino acid type (of 1'1g) -sugar (from the polynucleotide).
It is understood that a person skilled in the art can select other compounds original biological having the characteristics required for use in the present invention, namely the possibility of creating multiple bonds with a molecule of interest (it may for example be nucleic acids), a form in tree structure ensuring excellent availability and increasing the density, and an absence of interference with the hybridization reaction.
For the implementation of the invention, the trunk of the molecule serving as an arm is fixed on the chip holder, preferably so covalent, and the target nucleic acids are attached (or synthesized directly) to the a piece of several or all the trees of the molecule, preferably way covalent. Figure 1 gives a spatial representation of a biochip constructed by means of such an arm, in comparison with the biochips manufactured with linear arms. Preferably, a unique type of molecules of arms is used on the same biochip, at a density which can be adapted by the man of career. However, it is understood that molecules of structure and / or different length and / or shape can be used on the same biochip.
The nucleic acids can, for example, be attached to the arm by through an amine group that is reactive to an attack nucleophile, whatever the nature of this attack. For example, an amine group can be covalently linked to a nucleic acid by the action of a exposure to light. More generally, the polymer arm contains preferentially, at the end of its arms, a chemical group activatable, whatever it is, which has the ability to be covalently linked with a phosphate group (or other) at the chain end of a nucleic acid. This group activatable chemical may be present during the synthesis of the polymer or to be added later. It can be active from the start during synthesis of polymer or when it is fixed on the polymer, or else be activated chemically in a second time.

WO 01/44503 CA 02406948 2002-06-14 pCT ~ R00 / 03572 A number of chemical modes of covalent bonding between a nucleotide or a nucleic acid on the one hand, and a support of nature no nucleic acid on the other hand, have been described in the prior art, for example for the synthesis of oligonucleotides in vitro or for the fixation of oligonucleotides on a 5 metal support. These techniques can be used as part of the present invention for fixing nucleic acids on a DNA chip, in particular by fixing them by means of arms as defined above.
This includes the possibility of carrying out this fixing by means of a type system streptavidin.
10 The arm can be attached to the support by means of a group activatable chemical, at the end of the trunk, which can interact with molecules of support so as to create a bond, ideally of the covalent bond type.
As indicated above, the use of tree arms according to the present

The invention offers many advantages in terms of acid density nucleic targets. For example, in the case of "spots" of 28 pm in diameter:
- In case "A" of the fixation of the targets on the support via of non-arborescent fixation arms (stochiometric molecular ratio arms / targets = 1), or the direct fixation of the targets on the support, the surface of presentation targets (surface to which the probes will access to be hybridized to targets) is: (rr) x (radius) 2 = (rr) x 8 2 = a (Nm2) - In case "B" of the fixation of the targets on the support via of tree arms, we can estimate that each arm forms a structure hemispherical at the target presentation surface (surface of fixation of the targets on the arm), that is, in other words, that the surface total of presentation of targets is to:
(1/2) x (4) x (~ rr) x (radius) 2 = 2 x (rr) x A 2 = 2 xa (pm2).
This therefore amounts to doubling the presentation area of case "A", for a constant target density. Or vice versa, for the same quantity of targets through "spot", to reduce their density by a factor of 2 and therefore to improve accessibility of targets for probes (reduction in steric hindrance). By extension, if the WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

16 the surface of the tree arms is oblong, the surface of presentation targets is further increased.
In addition, the density of fixation of targets on tree arms is, at a priori, all the stronger as that obtained by direct fixing of targets on the chip support or target fixation via arms linear (not tree-like) because, for the same number of targets set by "spot" of the chip, the steric hindrance of the arms themselves on the surface of the support of the chip is lower in the case of tree arms compared to the arms linear.
Therefore, in reality, the target density must be able to be increased of more than a factor of 2 according to this principle, or the steric hindrance of the targets must be reduced by more than a factor of 2.
This aspect of the invention therefore makes it possible, on the one hand, to increase the density of targets per chip spot (and therefore increase the detection sensitivity when measuring the hybridization of the probes on the targets), and on the other hand simultaneously to decrease the steric hindrance of targets (and therefore to favor the hybridization of probes on targets, which also increases detection sensitivity).
As indicated above, biochips comprising this type of arm can be composed of a support comprising a flat and / or curved surface, solid or semi-solid nature. In addition, these supports may include materials such as glass, silica, poly-lysine, amino-silanes and / or silanes amino-reagents, or negatively charged carriers, as will be described in details in the rest of the text. On the other hand, immobilized target nucleic acids on the arm trees can also be of a nature, composition and of various origins. So it can be single or double nucleic acids strand, of natural, synthetic and / or semi-synthetic origin. In particular, it can be synthetic oligonucleotides, PCR products, genes or fragments of genes, plasmids (or other vectors such as cosmids, YAC, phages, etc.), single or double-stranded cDNA or RNA. Likewise, the length of the acids nucleic acids can be varied. However, it is preferred, however, that the acids WO 01/44503 CA 02406948 2002-06-14 pCT ~ R00 / 03572

17 nucleic acids are less than about 1000 bases in length (or pairs of basics).
In a particular embodiment, the target nucleic acids consist of single-stranded nucleic acids, including nucleic acids single-strand having a length less than 1000 bases.
In a particular embodiment, the target nucleic acids consist of single-stranded RNA, preferably having a shorter length at 1000 bases. It can in particular be total RNA or mRNA, taken from of a biological sample. The present invention indeed describes for the first times the use of single-stranded RNA as targets on a biochip. This constitutes another particular object of the present application.
In another variant of the invention, it is single-stranded DNA, or mixtures of single-stranded DNA and RNA.
In a preferred embodiment of the invention, the acids single-stranded target nucleic acids are between 25 and 100 nucleotides approximately, preferably between 30 and 60 nucleotides approximately.
In this regard, a particular object of the present invention also resides in a biochip composed of single-stranded nucleic acids attached to a support, single-stranded nucleic acids having a length of between 25 and 100 approximately nucleotides, preferably between 30 and 60 nucleotides. The invention resides also in the use, as target nucleic acids, of a population (of fragments) of single-stranded nucleic acids having a length between and approximately 60 nucleotides, designated in the present application by the expression "long oligonucleotides".
According to a first particular variant, the single-stranded nucleic acids are single-stranded DNA between 30 and 60 nucleotides in length about.

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18 According to another particular variant, the single-stranded nucleic acids are single-stranded RNAs of length between approximately 30 and 60 nucleotides.
Advantageously, these are nucleic acids produced by synthesis chemical in vitro, according to techniques known to those skilled in the art (synthesizers). These oligonucleotides are then, in a second step immobilized on the support.
The use (of fragments) of nucleic acids of such length allows, by opposition to the products currently available, to obtain a hybridization specific probes on their corresponding targets on biochips. In effect, as previously indicated, the use of oligonucleotides 25 bases long or less, as described in the prior art, results in the presence of many non-specific hybridizations of the probes, and therefore poor reproducibility of experiments using this system. Use according to the invention longer oligonucleotides, especially with a length between 30 and nucleotides, can on the contrary make it possible to obtain an absolute specificity hybridization.
The long oligonucleotides can, once synthesized, be fixed on the biochip support, neatly. This fixation can be either direct (fixation of the long oligonucleotide directly on the support), or indirect (fixation covalent of the long oligonucleotide on an "arm" as defined and described above before this arm being fixed on the support). The advantage of fixing the oligonucleotide along way indirect is to increase the accessibility of the probes to the sequence of the oligonucleotide, compared to the direct fixation situation where clutter steric is more important. However, the direct fixation of the long oligonucleotide is possible and can allow sufficient specific hybridization for reproducible use of oligonucleotide biochips. Regardless fashion of fixation (direct or indirect), the oligonucleotides are fixed by one of their ends (5 'or 3') and not over their entire length, to give probe one good accessibility to oligonucleotides. Furthermore, in the case of a fixation indirect, two choices are possible: either the long oligonucleotides are set by one of their ends on the arm, and the oligonucleotide-arm complexes are WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572

19 then deposited in an orderly fashion on the support and fixed to it, i.e.
arm is first attached to the support, and the long oligonucleotides are then deposited from orderly on the arm-support complex and attached to the arms.
In this embodiment, the type of support and arms used can be as described above. The use of this type of biochips oligonucleotides long chemically synthesized according to the present invention provides certain advantages over using shorter single strand targets and / or double strand and longer targets produced by PCR
First of all, the fact that the target is of single-strand strike allows to improve the yield of the probe hybridization reaction on the target. Indeed, if the target is double stranded, a significant number of targets will hybridize sure themselves during this reaction (the second strand of the target is in competition with the probe for hybridization).
Then, the direct chemical synthesis of the targets is simpler to manage as the synthesis of targets by PCR. In the first case, only knowledge sequences of targets that we want to have on the chip is necessary, so that in the second case it is necessary to have materially acids nucleic acids containing these sequences (generally in the form of clones) in order to practice their specific amplification by PCR.
On the other hand, PCR amplification produces a population of acids heterogeneous double strand nucleic acid with, in the vast majority of cases, presence of contaminating fragments, even if these fragments are in the minority in the final product of the reaction. This leads to the presence of hybridizations non-specific target probes and therefore background noise during measured hybridization rates. Conversely, direct synthesis of simple targets strand allows obtain nucleic acid fragments of very homogeneous sequence and allows in the long term to obtain purer targets and consequently less hybridization aspecific target probe. This specificity is all the more guaranteed when the biochips have arms, tree or not, electrically charged as will be described later.

WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572 Finally, the chemical synthesis of single-strand probes can be automated from so as to obtain purified targets, whereas the double-stranded targets produced by PCR must be purified before their fixation on the support.
5 The biochips with long synthetic oligonucleotides according to the invention therefore represent a significant technical advantage over the products and to the processes described in the prior art. This advantage is all the greater when these biochips have tree arms or loaded arms or supports electrically.
Indeed, the present invention further describes new approaches for control the spatial arrangement of nucleic acids and / or arms on the biochip, so as to favor the conditions of presentation, and therefore of hybridization of target probes. In particular, the present invention now shows that it is possible to determine (and modify) the electrical characteristics of arms (these are organic polymers of protein, carbohydrate nature or lipid, or synthetic inorganic polymers) or biochip, so as to improve the hybridization conditions, in particular the selectivity and the sensitivity of biochips.
This aspect of the invention can be implemented on all types possible oligonucleotide attachment arms on biochip supports, than their primary structure is linear or tree-like, and whatever the nature of chip support.
Thus, it is known that nucleic acids are negatively charged (from fact that these are weak acids which are, under usual conditions, in environments with a pH greater than their pKa; their acid functions are located on the phosphate groups which provide the bond between the sugars of nucleotides). Interactions between nucleic acid sequences can to be, in simplification, schematized as follows: on the one hand, these sequences can be hybridized with each other by establishing hydrogen bonds and strengths of Van der Waals between complementary bases (which can be considered WO 01/44503 CA 02406948 2002-06-14 pCT ~ R00 / 03572 as an affinity between them), on the other hand they can repel themselves from the made of their negative charges at the phosphate group level. To get a stable hybridization between two single stranded nucleic acid molecules it must the attraction linked to the hydrogen and Van der Waals bonds is greater than the repulsion related to negative charges, which is the case when the number of basics complementary adjacent is enough to get an attraction better than repulsion. Another parameter that comes into account is that of constraints atomic folding of molecules (secondary and tertiary structures).
These general principles regarding nucleic acids are also valid within of the same single-stranded nucleic acid molecule, whatever its length. They explain the capacity or not of such a molecule to hybridize or no on herself. Regarding the target attachment arms on the support of the chip, they too are subject to atomic structural constraints secondary and steric hindrance, but they do not exhibit any phenomenon attraction and hybridization such as those described for nucleic acids.
These stresses on the arms are also influenced by the fact that strands nucleic acids are attached to their) end (s).
One of the problems posed by the manufacturing of biochips, that targets fixed are single-stranded oligonucleotides (regardless of their length) or some Double-stranded DNA (whether whole plasmids or products of reaction of chain polymerization (PCR)), is that of the accessibility of these targets to the probe molecules that we want to analyze and that we want to hybridize in a way specific on their corresponding targets. This accessibility conditions the yield of hybridization reactions and therefore the sensitivity and specificity of probe analysis. This accessibility to probe hybridization-target is reduced in particular by the steric hindrance of the targets fixed on the chip, by the interactions between targets located close to each other in reason steric hindrance (partial hybridizations) and by structures secondary of these (intramolecular folding of the targets). The difficulties linked to steric hindrance are generated by the need to arrange the targets with a high density on each "spot" of the chip if you want power detect low levels of specific nucleic acids (sensitivity of detection) and WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572 reduce the saturation effects of hybridization capacities which risk hinder quantification of hybridization signals. This is why a principle general is to deposit molecular quantities of targets such that they are in large excess compared to the corresponding molecules of the probes.
The present invention overcomes these drawbacks. She describes in effect the use of special arms or supports, reducing interference between the targets, and thus ensuring better hybridization, including understood when targets are present at high densities. More particularly the present application shows that it is possible to use, to set targets on the biochip support, fixing arms with electric charge negative or negatively charged media.
Another object of the invention thus lies in a biochip comprising nucleic acids immobilized on a support by means of arms carrying a charge negative electric. Another object of the invention resides in a biochip comprising nucleic acids immobilized on a support carrying a charge electric negative uniform The present invention also resides in the use of molecules negatively charged or negatively charged media, for preparation biochips.
The use of fixing arms which are negatively charged so uniform, under the experimental conditions of hybridization of the probes on the targets, advantageously provides the following phenomena:
- The arms exert repulsive electrical forces towards the arms adjacent, which has the effect of reducing both their folds clean and their steric hindrance, especially at their ends set on targets;
- The arms exert repulsive electrical forces towards targets which are fixed at their ends, which has the effect of pushing the targets to move outwards, that is to say away from the support of the chip, the making it more accessible to probes;

WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572 - These mutual repulsion constraints have the consequence of limiting the attractive interactions between adjacent probes by moving them away from each other others, making them more accessible to probes and reducing risks mutual attractions linked to Van der Waals forces or connections hydrogen;
- This negative electric field exerted on the side of the targets attached to the arms has tendency to undo secondary structures of targets (i.e.
linearize) and therefore decrease the folding of targets on them-same, making them more accessible to probes;
- The layer located near the chip holder and formed by the arms is generally negatively charged, which has the effect of exerting a electrical repulsion force with respect to the probes (themselves charged negatively) and therefore to reduce the phenomena of non-specific hybridizations target probes. This makes it possible to reduce the background noise phenomena during analysis of hybridization results, and therefore increase both the sensitivity and specificity of the analyzes carried out by the technology of chips to DNA.
This particular aspect of the invention therefore provides an improvement clear technological methods, ensuring better selectivity and sensitivity to biochips.
Of course, this aspect of the invention is not limited to biochips in which arms are used. On the contrary, it also applies to use of a uniformly charged negatively charged chip carrier, within situations where the targets are directly fixed on the support without intermediate arms.
This is valid that the establishment of negative charges on the support is performed before or after fixing the targets on the support. This is a principle different from that of depositing the targets on micro-electrodes to which an electric current can be applied, because in the latter case the electrical charge of the support is not homogeneous.

WO 01/44503 CA 02406948 2002-06-14 pCT / FR00 / 03572 In this aspect of the invention, the biochips can comprise any type of target nucleic acid population as defined above, and the arms negatively charged can also have a tree form, as mentioned above.
The present invention also resides in a process for the preparation of biochips, as described above, comprising the immobilization (or the fixation) nucleic acids on a support by means of tree-shaped arms comprising a polymer of biological origin.
The preparation process can include a first step of fixing the arms tree-shaped on the support, followed by a second step of fixing of the target molecule in the tree-shaped arm. According to another mode of embodiment of the present invention, the method for preparing the biochip can understand a first step of fixing the target molecule to the arm by form tree structure then a second step of fixing the complex, obtained during the first step, to support.
The present invention also resides in the use of biochips such as described above, for experimental, therapeutic and diagnostic purposes. In in particular, the invention relates to the use of the biochips described above - to study the regulation of gene expression, or - for the search for genes or gene fragments, or - for target identification, or - for genetic diagnosis.
In these applications, the biochips are brought into contact with a population of “probe” nucleic acids, generally previously labeled, then the hybridization profile of the probes on the biochip is determined, according to the techniques known to those skilled in the art. An object of the invention therefore resides also in a method for analyzing polynucleotides, comprising setting up contact of a population of polynucleotides, preferably labeled, with a biochip according to the invention, and the demonstration of the formation of hybrids.

WO 01/44503 CA 02406948 2002-06-14 pCT ~ R00 / 03572 It is understood that the present invention is not limited to the modes specific embodiments described above, but also extends to variants of execution entering the normal knowledge of the skilled person.

Claims (30)

1. Biochip, characterized in that it comprises nucleic acids immobilized on a support by means of tree-shaped arms comprising a polymer of biological origin. 2. Biochip according to claim 1, characterized in that the arm is a polymer branched, having one or more levels of branching. 3. Biochip according to claim 1 or 2, characterized in that the arm is a organic polymer. 4. Biochip according to one of claims 1 to 3, characterized in that the east arm a polymer of sugars, for example glycogen or a derivative of glycogen or amylopectin. 5. Biochip according to one of claims 1 to 3, characterized in that the east arm a branched polymer comprising a repeat of galactose monomers, glucose, mannose, fucose, xylose, N-acetylgalactosamine and/or N-acetylglucosamine. 6. Biochip according to one of claims 1 to 3, characterized in that the east arm a glycopolypeptide, for example aggrecan. 7. Biochip according to one of claims 1 to 3, characterized in that the east arm a polypeptide compound, for example an immunoglobulin. 8. Biochip according to any one of the preceding claims, characterized in that nucleic acids are covalently attached to the ends of the arm tree. 9. Biochip according to any one of the preceding claims, characterized in that the arms are attached to the support, covalently, by the part trunk of the molecule. 10. Biochip according to any one of the preceding claims, characterized in that the support has a flat and/or curved surface. 11. Biochip according to claim 10, characterized in that the support is a solid or semi-solid support. 12. Biochip according to claim 11, characterized in that the support is composed of glass, silica, poly-lysine, amino-silanes and/or amino-silanes reagents. 13. Biochip according to any one of the preceding claims, characterized in that the nucleic acids are single or double nucleic acids strand, of natural, synthetic and/or semi-synthetic origin. 14. Biochip according to claim 13, characterized in that the acids nucleic are chosen from synthetic oligonucleotides, PCR products, Genoa or gene fragments, plasmids. single- or double-stranded cDNAs or RNA. 15. Biochip according to claim 14, characterized in that the acids nucleic are single-stranded nucleic acids, including acid molecules single-stranded nuclei of length between 25 and 100 nucleotides about, preferably between 30 and 60 nucleotides approximately. 16. Use of a polymer of sugars derived from glycogen having a tree-like spatial organization, for binding acids nucleic on supports.

28~ 17. Biochip according to claim 13, characterized in that the acids nucleic are single-stranded nucleic acids with a length between 30 and nucleotides approx. 18. Biochip according to claim 17, characterized in that the acids nucleic single-stranded are single-stranded DNAs of length between 30 and 60 nucleotides approx. 19. Biochip according to claim 17, characterized in that the acids nucleic single-stranded are single-stranded RNAs of length between 30 and 60 nucleotides approx. 20. Biochip according to one of claims 17 to 19, characterized in that the single-stranded nucleic acids are nucleic acids produced by synthesis chemical in vitro. 21. Biochip according to one of claims 17 to 20, characterized in that it comprises RNAs immobilized on a solid or semi-solid support by means of tree-shaped arm comprising a polymer of biological origin. 22. Biochip according to one of claims 17 to 20, characterized in that it comprises PCR product nucleic acids immobilized on a support solid or semi-solid by means of tree-shaped arms comprising a polymer of biological origin. 23. Biochip according to any one of claims 1 to 15 and 17 to 22, characterized in that the arm carries a negative electrical charge. 24. Biochip according to any one of claims 1 to 15 and 17 to 22, characterized in that the support bears a negative electric charge uniform. 25. Use of a biochip according to one of claims 1 to 15 and 17 to 24, for study the regulation of gene expression. 26. Use of a biochip according to one of claims 1 to 15 and 17 to 24, for the search for genes or gene fragments. 27. Use of a biochip according to one of claims 1 to 15 and 17 to 24, for target identification. 28. Use of a biochip according to one of claims 1 to 15 and 17 to 24, for genetic diagnosis. 29. Process for preparing a biochip according to one of claims 1 to 15 and 17 to 24, characterized in that it comprises:
a) a step of fixing the tree-shaped arm on the support, and, b) a step of attaching the target molecule to the tree-shaped arm obtained in a). 30. Process for the preparation of a biochip according to one of claims 1 to 15 and 17 to 24, characterized in that it comprises:
a) a step of fixing the target molecule to the tree-shaped arm, b) a step of fixing the complex obtained in a) on the support.