U.S. patent application number 17/834197 was filed with the patent office on 2022-09-22 for method for identifying high-affinity complexes made of two ligands and one receptor, device for implementing the method and self-assembling chemical library for use in the method.
This patent application is currently assigned to DYNABIND GMBH. The applicant listed for this patent is DYNABIND GMBH. Invention is credited to Helena DE ANDRADE, Weilin LIN, Francesco REDDAVIDE, Yixin ZHANG.
Application Number | 20220298552 17/834197 |
Document ID | / |
Family ID | 1000006388062 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298552 |
Kind Code |
A1 |
REDDAVIDE; Francesco ; et
al. |
September 22, 2022 |
METHOD FOR IDENTIFYING HIGH-AFFINITY COMPLEXES MADE OF TWO LIGANDS
AND ONE RECEPTOR, DEVICE FOR IMPLEMENTING THE METHOD AND
SELF-ASSEMBLING CHEMICAL LIBRARY FOR USE IN THE METHOD
Abstract
The present invention relates to a method for the sensitive
identification of high-affinity complexes made of two ligands (2,
3, 4, 5, 6, 7) and one receptor (1). A large number of different
ligands (2, 3, 4, 5, 6, 7) of a chemical library are hereby
contacted with at least one receptor (1) in a solution. The ligands
of the library have a single-strand DNA (8, 9) or RNA with a base
length of 2 to 10 bases or alternatively more than 10 bases. In
addition, the solution is incubated for a specific period of time
and complexes made of two ligands (2, 3, 4, 5, 6, 7) and one
receptor (1) are identified.
Inventors: |
REDDAVIDE; Francesco;
(Dresden, DE) ; DE ANDRADE; Helena; (Dresden,
DE) ; LIN; Weilin; (Dresden, DE) ; ZHANG;
Yixin; (Dresden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DYNABIND GMBH |
Dresden |
|
DE |
|
|
Assignee: |
DYNABIND GMBH
Dresden
DE
|
Family ID: |
1000006388062 |
Appl. No.: |
17/834197 |
Filed: |
June 7, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15326172 |
Jan 13, 2017 |
11365439 |
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PCT/EP2015/065874 |
Jul 10, 2015 |
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17834197 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6811 20130101;
B01L 2300/1894 20130101; B01L 3/5027 20130101; B01L 2300/088
20130101; B01L 7/525 20130101; G01N 33/5308 20130101; B01L
2300/1861 20130101; B01L 2400/0487 20130101; B01L 2300/0816
20130101; B01L 2300/1822 20130101; B01L 2300/0681 20130101 |
International
Class: |
C12Q 1/6811 20060101
C12Q001/6811; B01L 3/00 20060101 B01L003/00; B01L 7/00 20060101
B01L007/00; G01N 33/53 20060101 G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2014 |
DE |
10 2014 213 783.7 |
Claims
1-29. (canceled)
30. A method for sensitive identification of high-affinity
complexes made of two ligands and one receptor, comprising the
steps of: i) interaction of a large number of different complexes
made of ligands of a chemical library with at least one receptor in
a solution, the ligands of the library having a single-strand DNA
or RNA with a base length of more than 2 bases, which is bonded
chemically covalently to the ligands, and only 2 to 10 bases of the
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands, and the ligands being complexed to form ligand
complexes via hybridisation of the DNA or RNA; ii) incubation of
the solution for a specific period of time, complexes made of
ligand complexes and the receptor being produced; iii)
identification of the resulting complexes made of ligand complexes
and the receptor, wherein the solution is incubated at a
temperature at which, between the first part of the ligands and the
second part of the ligands, an equilibrium of hybridisation to form
ligand complexes and dissociation to form free ligands arises.
31. The method according to claim 30, wherein only 3 to 9 bases of
the single-strand DNA or RNA of a first part of the ligands are
complementary to the single-strand DNA or RNA of a second part of
the ligands.
32. The method according to claim 30, wherein the length of the DNA
or RNA of the first part of the ligands and/or the length of the
DNA or RNA of the second part of the ligands is at least 20
bases.
33. The method according to claim 30, wherein the first part of the
ligands has L different ligands and the second part of the ligands
has M different ligands so that L M difference ligand complexes are
formed.
34. The method according to claim 30, wherein the solution is
incubated in step ii) at a temperature of 1.degree. C. to
50.degree. C.
35. The method according to claim 30, wherein the solution is
incubated in step ii) for a period of time of 0.1 to 48 hours.
36. The method according to claim 30, wherein the receptor is
immobilized on a substrate selected from the group consisting of
glass, ceramic, biopolymer, sepharaose, synthetic polymer and
hydrogel.
37. The method according to claim 30, wherein the receptor
comprises or consists of a protein, a DNA, a RNA, a cell and/or an
organic molecule with a molecular mass .ltoreq.200 kDa.
38. The method according to claim 30, wherein the ligand comprise
or consist of a molecule selected from the group consisting of
protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and
ssRNA, aptamer, organic molecule with a molecular mass .ltoreq.200
kDa.
39. The method according to claim 30, wherein the complexes made of
ligand complexes and the receptor are identified via an analytical
method selected from the group consisting of mass spectrometry,
HPLC, gas chromatography, IR spectroscopy and DNA sequencing of a
DNA or RNA barcode.
40. The method according to claim 30, wherein the single-strand DNA
or RNA in the first part of the ligands and/or the single strand
DNA or RNA in the second part of the ligands comprises a base
sequence which codes for the chemically covalently-bonded
ligands.
41. The method according to claim 30, wherein the single-strand DNA
or RNA of the ligands of the library is hybridized in regions to
form a complementary base sequence of a further single-stranded DNA
or RNA, 2 to bases on the further single-strand DNA or RNA of a
first part of the ligands being complementary to bases of the
single-strand DNA or RNA of a second part of the ligands and
binding to the complementary bases, during the method, a Y-form
being configured, and a ligase being added during the method, which
ligates chemically covalently the further single-strand DNA or RNA
of the first part of the ligands to the further single-strand DNA
or RNA of the second part of the ligands.
42. The method according to claim 41, wherein, in the case of a
single-strand DNA or RNA of a first part of the ligands, i) which
has the ligand at the 5' end thereof, has the bases which are
complementary to the bases of the single-strand DNA or RNA of a
second part of the ligands, at the 5' end thereof, and the 2 to 10
bases of the further single-strand DNA or RNA, which are
complementary to bases of the single-stranded DNA or RNA of a
second part of the ligands, are disposed at the 3' end of the
further single-strand DNA or RNA; or ii) which has the ligand at
the 3' end thereof, has the bases, which are complementary to the
bases of the single-strand DNA or RNA of a second part of the
ligands, at the 3' end thereof, and the 2 to 10 bases of the
further single-strand DNA or RNA, which are complementary to bases
of the single-strand DNA or RNA of a second part of the ligands,
are disposed at the 5' end of the further single-strand DNA or
RNA.
43. A chemical library, comprising ligands which have bonded
chemically covalently a single-strand DNA or RNA with a base length
of more than 2 bases, wherein only 2 to 10 bases of the
single-strand DNA or RNA of a first part of the ligands are
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands.
44. The library according to claim 43, wherein only 3 to 9 bases of
the single-stranded DNA or RNA of a first part of the ligands are
complementary to the single-strand DNA or RNA of a second part of
the ligands.
45. The library according to claim 43, wherein the length of the
DNA or RNA of the first part of the ligands and/or the length of
the DNA or RNA of the second part of the ligands is at least 20
bases.
46. The library according to claim 43, wherein the first part of
the ligands has L different ligands and the second part of the
ligands has M different ligands so that the library has LM
different ligand complexes.
47. The library according to claim 43, wherein the ligands comprise
or consist of a molecule selected from the group consisting of
protein, peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and
ssRNA, aptamer, organic molecule with a molecular mass .ltoreq.200
kDa.
48. The library according to claim 43, wherein the single-strand
DNA or RNA of the ligands of the library is hybridised respectively
in regions to form a complementary base sequence of a further
single-strand DNA or RNA, 2 to 10 bases of a first further
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the second single-strand DNA or RNA of a
second part of the ligands.
49. The library according to claim 48, wherein the single-strand
DNA or RNA of a first part of the ligands, i) which has the ligand
at the 5' end thereof, has the bases, which are complementary to
the bases of the single-strand DNA or RNA of a second part of the
ligands, at the 5' end thereof, and the 2 to 10 bases of the
further single-strand DNA or RNA, which are complementary to bases
of the second single-strand DNA or RNA of a second part of the
ligands, are disposed at the 3' end of the further single-strand
DNA or RNA; or ii) which has the ligand at the 3' end thereof, has
the bases, which are complementary to the bases of the
single-strand DNA or RNA of a second part of the ligands, at the 3'
end thereof, and the 2 to 10 bases of the further single-strand DNA
or RNA, which are complementary to bases of the single-strand DNA
or RNA of a second part of the ligands, are disposed at the 5' end
of the further single-strand DNA or RNA.
50. A method in which the library according to claim 43 is used for
selective and sensitive identification of high-affinity ternary
ligand-receptor complexes.
Description
[0001] A method for identifying high-affinity complexes made of two
ligands and one receptor is proposed. In the method, ligands of a
library which have respectively a single-strand DNA or RNA are
used. Since the DNA or RNA of one part of the ligands is
complementary to the DNA or RNA of another part of the ligands,
both parts of the ligands together can form binary complexes. The
length of the DNA or RNA, in one embodiment, can be 3 to 10 bases
or, in another embodiment, more than 10 bases. According to the
invention, hybridisation and dissociation of the single-strand DNAs
or RNAs is provided in a dynamic manner which is achieved in fact
at room temperature for the first embodiment and, for the second
embodiment, a cycle of heating and cooling is required.
Furthermore, a microfluidic device for implementing the method
according to the invention and a dynamic, self-assembling chemical
library comprising binary complexes of the respective ligands is
proposed.
[0002] An essential requirement in the use of DNA-coded chemical
libraries and DNA/RNA aptamer libraries for finding effective
binders and inhibitors relative to various target proteins of
biomedical interest is improving the signal-to-noise ratio in
selection experiments in order to distinguish molecules with high
binding affinity from weak or non-binding compounds.
[0003] To date, in the prior art generally parallel selection
experiments with differing selection stringency have been
implemented in order to optimise the selection conditions relative
to a known target protein. For example solid carrier materials were
thus charged with different quantities of the target protein.
Furthermore, various washing methods were established, in which
buffers with different ion strengths and detergents were used.
[0004] In order to implement parallel selection experiments, many
additional experiments for a target protein must be implemented.
This is both time-consuming and expensive. In addition, it is very
difficult to design parallel selection experiments with varying
stringency in the selection if the distributions of the binding
affinities of the entire library for the target protein are
unknown. In order to select binders with high affinity, special
selection conditions must be applied. Accordingly, it is a
disadvantage that a stringent, parallel selection experiment can
only be designed after it is known that high-affinity binding
molecules are located in the library.
[0005] On the other hand, also the characterisation of components
of a first selection experiment (de novo selection experiment) is
very time-consuming and costly. In addition, it is difficult to
design a parallel selection experiment without the knowledge and
the information resulting from a preceding primary selection.
[0006] The object of the present invention is hence the provision
of a method with which, in a simple, economical and
highly-sensitive manner, identification of high-affinity ternary
complexes made of two ligands and one receptor is possible. In
addition, a device for implementing the method and a
self-assembling chemical library for use in the method should be
provided.
[0007] The object is achieved as described below by the method
according to the invention, the dynamic self-assembling chemical
library according to the invention and the use of the library
according to the invention.
[0008] According to the invention, a method for sensitive
identification of high-affinity complexes made of two ligands and
one receptor is provided, which comprises the following steps:
[0009] a) interaction of a large number of different complexes made
of ligands of a chemical library with at least one receptor in a
solution, the ligands of the library having a single-strand DNA or
RNA with a base length of more than 10 bases, which is bonded
chemically covalently to the ligands, and more than 10 bases of the
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands, and the ligands being complexed to form ligand
complexes via hybridisation of the DNA or RNA; [0010] b) incubation
of the solution for a specific period of time, complexes made of
ligand complexes and the receptor being produced; [0011] c)
dissociation of the ligand complexes to form free ligands; [0012]
d) re-hybridisation of the free ligands to form further ligand
complexes; [0013] e) incubation of the solution for a specific
period of time, further complexes made of the further ligand
complexes and the receptor being produced; [0014] f) identification
of the resulting complexes made of ligand complexes and the
receptor.
[0015] The method is characterised in that incubation takes place
at a temperature (step b), step d) and/or step e)) at which the
first part of the ligands and the second part of the ligands are
hybridised in the solution to form ligand complexes.
[0016] In the presence of a receptor which has high-affinity to a
specific combination of two ligands (specific binary ligand complex
made of ligand 1 and ligand 2 via dsDNA or dsRNA), a high-affinity
ternary ligand-receptor complex made of the two ligands and the
receptor is formed. High-affinity ligand pairs are hence depleted
in the pool of binary ligand pairs and enriched in the form of a
ternary ligand pair-receptor complex.
[0017] The disadvantage of methods from the state of the art is
that a specific percentage of high-affinity ligands with few as far
as absolutely no affine ligands is hybridised and hence is
"trapped" in low-affinity ligand complexes. This problem is
resolved by the method according to the invention. By means of a
method step in which ligand pairs are specifically dissociated, the
high-affinity ligands "trapped" previously in the ligand complex
become "free" again in order to hybridise, in a further
hybridisation step of the method, with other free high-affinity
ligands to form new ligand complexes. The newly formed ligand
complexes are in turn available for forming ligand-receptor
complexes. As a result, the high-affinity ligands--in the ideal
case quantitatively--can hence be specifically converted into
ligand-receptor complexes, which produces a larger quantity of
ligand-receptor complexes and hence increases the sensitivity of
the method relative to methods from the state of the art (improved
signal-to-noise ratio). In fact, the frequency of the formation of
the high-affinity ligand pair, in the case of a number of N
theoretically possible high-affinity ligand pairs in a library, can
be increased ideally up to a factor N (quantitative formation of
high-affinity complexes, no high-affinity ligands are bonded to
low-affinity ligands and hence "trapped").
[0018] The ligand complexes can be dissociated for example by
increasing the temperature of the solution, in particular to a
temperature of 35.degree. C. to 95.degree. C., preferably
50.degree. C. to 95.degree. C., particularly preferably 70.degree.
C. to 95.degree. C., in particular 80.degree. C. to 95.degree.
C.
[0019] The dissociation of the ligand complexes is preferably
implemented in a part of the solution which is spatially at a
spacing from the receptor, preferably such that the receptor is not
functionally impaired by the conditions which lead to the
dissociation. The advantage of this embodiment is that the effect
of the dissociation does not have a negative effect on the
(biological) function of the receptor. In particular, receptors are
consequently protected from thermal and/or pH-induced denaturation
(e.g. receptors which comprise proteins or consist thereof).
[0020] The free ligands can (again) be hybridised by lowering the
temperature of the solution, in particular by lowering to a
temperature of 1.degree. C. to 30.degree. C., preferably 5.degree.
C. to 28.degree. C., particularly preferably 10.degree. C. to
25.degree. C., in particular 15.degree. C. to 20.degree. C.
[0021] Steps c) to e) of the method can be repeated at least once
or twice, preferably at least 3 or 4 times, particular preferably
at least 5 or 6 times, in particular at least 7 or 8 times. The
more often the steps are repeated, the more are high-affinity
ligand complexes gradually converted into the ligand-receptor
complex (rise in sensitivity).
[0022] In a preferred embodiment, at least 20 bases, preferably at
least 40 bases, particularly preferably a least 100 bases, in
particular at least 200 bases, of the single-strand DNA or RNA of a
first part of the ligands are complementary to the single-strand
DNA or RNA of a second part of the ligands.
[0023] Furthermore, an alternative solution to the object according
to the invention is provided, which solution is possible without
heating and cooling steps.
[0024] In this respect, a method for sensitive identification of
high-affinity complexes made of two ligands and one receptor is
provided, which comprises the following steps: [0025] i)
interaction of a large number of different complexes made of
ligands of a chemical library with at least one receptor in a
solution, the ligands of the library having a single-strand DNA or
RNA with a base length of more than 2 bases, which is bonded
chemically covalently to the ligands, and only 2 to 10 bases of the
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands, and the ligands being complexed to form ligand
complexes via hybridisation of the DNA or RNA; [0026] ii)
incubation of the solution for a specific period of time, complexes
made of ligand complexes and the receptor being produced; and
[0027] iii) identification of the resulting complexes made of
ligand complexes and the receptor.
[0028] The method is characterised in that the solution is
incubated at a temperature at which, between the first part of the
ligands and the second part of the ligands, an equilibrium of
hybridisation to form ligand complexes and dissociation to form
free ligands arises (e.g. room temperature, i.e. 15.degree. C. to
30.degree. C.). As a result, a dynamic hybridisation and
dissociation is obtained.
[0029] The difference from the first-mentioned method according to
the invention is that, by means of the lower number of
complementary bases between the two parts of the ligands,
association (via hybridisation) and dissociation of the ligands
takes place in fact at room temperature (dynamic equilibrium). By
means of the presence of a receptor, high-affinity ligand pairs are
"withdrawn" from the dynamic equilibrium and converted into the
ligand-receptor complex. High-affinity ligand pairs are therefore
depleted in the general ligand pool of the chemical library and the
high-affinity, ternary ligand-receptor complex is enriched. By
means of the dynamic equilibrium, all high-affinity ligand pairs
(in the ideal case quantitatively) are therefore converted into the
receptor complex even without heating and cooling, which increases
the concentration of ligand-receptor complexes and hence improves
the sensitivity of the detection method. The disadvantage however
is an overall somewhat low affinity of the ligand pairs for the
receptor compared with the ligand pairs from the first-mentioned
method since the stability of the ligand pairs is somewhat less due
to the lower number of complementary bases and hence, in
equilibrium, also the ternary ligand-receptor complex is populated
less highly.
[0030] In this variant, only 3 to 9 bases, preferably only 4 to 8
bases, particularly preferably only 5 to 7 bases, of the
single-strand DNA or RNA of a first part of the ligands can be
complementary to the single-strand DNA or RNA of a second part of
the ligands.
[0031] In both methods, the length of the DNA or RNA of the first
part of the ligands and/or second part of the ligands can be at
least 20 bases, preferably at least 40 bases, particularly
preferably at least 100 bases, in particular at least 200
bases.
[0032] The first and/or second part of the ligands can constitute
more than 20%, preferably more than 30%, particularly preferably
more than 40%, in particular 50%, of the total quantity of the
ligands. The first part of the ligands preferably has L different
ligands and the second part of the ligands preferably has M
different ligands so that LM different binary ligand complexes are
formed. For a library with LM binary ligand complexes, a specific
high-affinity ligand complex can be enriched with increased
efficiency of at least 1.5 to N times, preferably 2 to N.sup.(1/2)
times, in particular 3 to 10 times.
[0033] In a preferred embodiment, the solution is incubated in at
least one of steps b), e) and ii) at a temperature of 1.degree. C.
to 50.degree. C. preferably 5.degree. C. to 37.degree. C.,
particularly preferably 10.degree. C. to 25.degree. C., in
particular 15.degree. C. to 20.degree. C. At these temperature
ranges, it is advantageous that many types of receptor (in
particular protein receptors) are stable here and have not lost
their function of binding ligands.
[0034] The solution can be incubated in at least one of steps b),
e) and ii) for a period of time of 0.1 to 48 hours, preferably 0.2
to 24 hours, particularly preferably 0.5 to 12 hours, in particular
1 to 6 hours. Fairly long incubation times have proved to be
advantageous since the binding of the ligand pairs to the receptor
can in fact be assisted thermodynamically but nevertheless can have
slow kinetics.
[0035] The receptor which is used in the methods according to the
invention can be immobilised, preferably on a substrate selected
from the group consisting of glass, ceramic, biopolymer, sepharose,
synthetic polymer and hydrogel. In particular, the solution with
ligands can hereby be guided past the immobilised receptor in a
cycle. This embodiment is advantageous if, in the method, the
dissociation of the ligand complexes is implemented in one part of
the solution which is spatially at a spacing from the receptor
since dissociation and hybridisation of the ligands can in fact be
separated hereby spatially but nevertheless take place at the same
time (e.g. dissociation at a distance from the receptor,
hybridisation close to the receptor).
[0036] The receptor preferably comprises a protein, a DNA, an RNA,
a cell and/or an organic molecule with a molecular mass 200 kDa,
preferably 100 kDa, particularly preferably 10 kDa, in particular 3
kDa, or consists thereof.
[0037] The ligands used can comprise a molecule selected from the
group consisting of protein, peptide, lipid, carbohydrate, dsDNA,
ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular
mass 200 kDa, preferably 100 kDa, particularly preferably 10 kDa,
in particular 3 kDa, or consist thereof.
[0038] In a preferred embodiment, the complexes made of ligand
complexes and the receptor are identified via an analytical method.
The method is preferably selected from the group consisting of mass
spectrometry, HPLC, gas chromatography, IR spectroscopy and DNA
sequencing, preferably DNA sequencing of a DNA- or RNA barcode.
[0039] Further preferably, the single-strand DNA or RNA in the
first and/or second part of the ligands comprises a base sequence
which codes for the chemically covalently-bonded ligand (base
sequence as barcode), this base sequence preferably being
hybridised to form a complementary base sequence of a further
single-strand DNA or RNA. The advantage of the barcode resides in
simple identification of complex-bonded ligands via DNA- or RNA
sequencing. It is particularly advantageous if the base sequence of
the ligands comprises a complementary single-strand DNA- or RNA,
hybridised, which can serve likewise as barcode. If both ligands of
the ligand-receptor complex (ligand 1 and 2) comprise a
single-strand DNA or RNA, then these can be ligated, e.g. by
addition of a ligase, a single-strand DNA- or RNA being obtained
which codes for the specific combination of ligand 1 and 2. The
(ligated) barcode in this case codes therefore not only for an
individual ligand but for two ligands which together form a
high-affinity ligand complex.
[0040] The single-strand DNA used in the method can comprise [0041]
i) adenine, thymine, guanine and/or cytosine; and/or
[0042] The single-strand RNA used in the method can comprise [0043]
ii) adenine, uracil, guanine, cytosine and/or inosine.
[0044] The method is characterised, in an advantageous embodiment,
in that the single-strand DNA or RNA of the ligands of the library
is hybridised in regions to form a complementary base sequence of a
further single-strand DNA or RNA, 2 to 10 bases of the further
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands and, during the method, binding to the
complementary bases (preferably forming a Y-shape, in particular as
illustrated in FIG. 6), a ligase being added during the method,
which ligase ligates chemically covalently the further
single-strand DNA or RNA of the first part of the ligands to the
further single-strand DNA or RNA of the second part of the
ligands.
[0045] A ligation of both further DNAs or RNAs hence takes places
only if the result is hybridisation of the respective bases, i.e.
if the total complex is sufficiently stable. It is advantageous
with this embodiment however that once formation of a complex has
taken place it is "stored" by the chemically covalent ligation of
both further DNAs or RNAs. In other words, the ligated further DNAs
or RNAs accumulate during the method if the basis for ligation
thereof has been stable formation of the total complex. In this
way, a clear increase in sensitivity of the method can be achieved,
especially if the accumulated, ligated DNA or RNA can be further
amplified via known methods (PCT, RT-PCR). As a result, also very
small concentrations of ligands can be used in this way or
high-affinity ligands can be identified even in the case of very
small receptor concentrations.
[0046] In the above-cited embodiment, in the case of a
single-strand DNA or RNA of a first part of the ligands, [0047] i)
which has bonded the ligand at the 5' end thereof, can have the
bases, which are complementary to the bases of the single-strand
DNA or RNA of a second part of the ligands, at the 5' end thereof,
and the 2 to 10 bases of the further single-strand DNA or RNA,
which are complementary to bases of the single-strand DNA or RNA of
a second part of the ligands, can be disposed at the 3' end of the
further single-strand DNA or RNA; or [0048] ii) which has bonded
the ligand at the 3' end thereof, can have the bases, which are
complementary to the bases of the single-strand DNA or RNA of a
second part of the ligands, at the 3' end thereof, and the 2 to 10
bases of the further single-strand DNA or RNA, which are
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands, are disposed at the 5' end of the further
single-strand DNA or RNA.
[0049] By means of this specific arrangement of the respective
complementary bases, the formation of a Y-structure is assisted,
which in turn increases the efficiency of the ligation of the two
further single-strand DNAs or RNAs by the ligase.
[0050] Furthermore, a microfluidic device for implementing the
method according to the invention is provided. The device
comprises: [0051] a) a container for receiving immobilised
receptor, the container having an access with valve which serves
for injection of ligands of a library and for isolation of
ligand-receptor complexes, and the container having an inlet and an
outlet for a fluid pipe; [0052] b) a fluid pipe which is connected
to an inlet and an outlet of the container and which has an outlet
with valve at one place; [0053] c) a heating device which is
disposed in a first region of the fluid pipe; and [0054] d) a
cooling region which is situated in a second region of the fluid
pipe, different from the first.
[0055] In the microfluidic device, the heating of ligands in
solution and the selection of ligands which are bonded to
immobilised receptor can take place locally separated from each
other and hence thermal damage to the receptor (e.g. protein
denaturation) can be prevented. The container can hereby retain
immobilised receptor and prevent immobilised receptor from passing
into the fluid pipes. Preferably, the container has hence filters
which prevent passage of immobilised receptor into the fluid
pipe.
[0056] In a preferred embodiment, the cooling region of the device
has a cooling device and/or is disposed in the region of the outlet
with valve.
[0057] The device can have a pump which is disposed such that it
can pump a liquid through the fluid pipe. In particular, the
container of the device can have filters which prevent passage of
immobilised receptor into the fluid pipe.
[0058] The microfluidic device can be characterised in that the
container and/or the fluid pipe comprises a dynamic,
self-assembling chemical library according to the invention (see
below).
[0059] Furthermore, a chemical library which comprises ligands is
provided, which ligands have a single-strand DNA or RNA with a base
length of more than 2 bases which are bonded chemically covalently
to a ligand. The library is characterised in that 2 to 10 bases of
the single-strand DNA or RNA of a first part of the ligands are
complementary to bases of the single-strand DNA or RNA of a second
part of the ligands.
[0060] Only 3 to 9 bases, preferably only 4 to 8 bases,
particularly preferably only 5 to 7 bases, of the single-strand DNA
or RNA of a first part of the ligands can hereby be complementary
to the single-strand DNA or RNA of a second part of the
ligands.
[0061] The length of the DNA or RNA of the first part of the
ligands and/or second part of the ligands can be at least 20 bases,
preferably at least 40 bases, particularly preferably at least 100
bases, in particular at least 200 bases.
[0062] In a preferred embodiment, the first and/or second part of
the ligands constitutes more than 20%, preferably more than 30%,
particularly preferably more than 40%, in particular 50%, of the
total quantity of the ligands of the library. The first part of the
ligands L preferably has L different ligands and the second part of
the ligands preferably has L different ligands so that the library
has L.sup.2 different ligand complexes.
[0063] The ligands of the library can comprise a molecule selected
from the group consisting of protein, peptide, lipid, carbohydrate,
dsDNA, ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a
molecular mass 200 kDa, preferably .ltoreq.100 kDa, particularly
preferably .ltoreq.10 kDa, in particular .ltoreq.3 kDa, or consist
thereof.
[0064] The single-strand [0065] i) DNA can comprise adenine,
thymine, guanine and/or cytosine; and/or [0066] ii) RNA can
comprise adenine, uracil, guanine, cytosine and/or inosine.
[0067] The library can be characterised in that the single-strand
DNA or RNA of the ligands of the library is hybridised respectively
in regions to form a complementary base sequence of a further
single-strand DNA or RNA, 2 to 10 bases of a first further
single-strand DNA or RNA of a first part of the ligands being
complementary to bases of the second single-strand DNA or RNA of a
second part of the ligands.
[0068] The single-strand DNA or RNA of a first part of the ligands,
[0069] i) which has the ligand at the 5' end thereof, can have the
bases which are complementary to the bases of the single-strand DNA
or RNA of a second part of the ligands, at the 5' end thereof, and
the 2 to 10 bases of the further single-strand DNA or RNA, which
are complementary to bases of the second single-strand DNA or RNA
of a second part of the ligands, are disposed at the 3' end of the
further single-strand DNA or RNA; or [0070] ii) which has the
ligand at the 3' end thereof, can have the bases which are
complementary to the bases of the single-strand DNA or RNA of a
second part, at the 3' end thereof, and the 2 to 10 bases of the
further single-strand DNA or RNA, which are complementary to bases
of the single-strand DNA (9) or RNA of a second part of the
ligands, can be disposed at the 5' end of the further single-strand
DNA or RNA.
[0071] Finally, the use of the chemical library according to the
invention for selective and sensitive identification of
high-affinity ternary ligand-receptor complexes is proposed.
[0072] The subject according to the invention is intended to be
explained in more detail with reference to the subsequent Figures
without wishing to restrict said subject to the specific
embodiments illustrated here.
[0073] FIG. 1 shows a large number of different ligands 2, 3, 4, 5,
6, 7 of a chemical library and at least one receptor 1. The ligands
2, 3, 4, 5, 6, 7 are bonded respectively chemically covalently to a
single-strand DNA 8, 9, the single-strand DNA 8, 9 having a base
length of more than 10 bases. The single-strand DNA 8, 9 codes
respectively for the respective ligand, to which it is bonded, via
the base sequence thereof. In this embodiment, 18 bases of the
single-strand DNA 8 of a first part of the ligands 2, 6, 7 are
complementary to bases of the single-strand DNA 9 of a second part
of the ligands 3, 4, 5 (see broken line between the single-strand
DNAs 8, 9). Here only a specific ligand pair (formed by the ligands
of the reference numbers 2 and 3) binds with high affinity to the
receptor 1. Other ligand pairs (formed by the ligands of the
reference numbers 4, 5, 6, 7) bind weakly as far as not at all to
the receptor 1. As a result of the high number of complementary
bases between the single-strand DNAs 8, 9 of two ligands 2, 3, 4,
5, 6, 7, the ligand complexes are stabilised, which causes high
sensitivity during use thereof in a detection method. In order to
achieve a dynamic of the hybridisation and dissociation, permanent
heating (dissociation) and cooling (re-association or
hybridisation), i.e. an energy supply, is necessary.
[0074] FIG. 2 shows an illustration corresponding to FIG. 1, with
the difference that, in this embodiment, only 6 bases of the
single-strand DNA of a first part of the ligands are complementary
to bases of the single-strand DNA or RNA of a second part of the
ligands (otherwise the molecules and references are identical to
FIG. 1). As a result of the lower number of complementary bases,
the advantage arises that the association (hybridisation) of two
ligands is highly dynamic already at room temperature (15.degree.
C. to 30.degree. C.), i.e. permanent hybridisation and dissociation
takes place. The high-affinity binding to receptor, present in the
case of specific ligand pairs, restricts the dissociation of these
ligand pairs, as a result of which complexes made of receptor and
high-affinity ligand pairs are populated for longer, "accumulate"
in the course of time and finally are populated more highly in the
equilibrium. It is advantageous in this embodiment that the dynamic
of the association and hybridisation takes place at room
temperature, i.e. in contrast to the embodiment of FIG. 1, no
thermal energy need be supplied. The disadvantage of this
embodiment is lower sensitivity than in the embodiment in FIG. 1
since the lower number of base pairs (6 instead of 18) makes the
ligand complex and hence the complex of ligands with the receptor
more unstable, as a result of which the latter is populated less
highly in the equilibrium than in the embodiment in FIG. 1.
[0075] FIG. 3 clarifies, via a reaction equation, how the
high-affinity ligand pairs "accumulate" as complex with the
receptor in the course of time (see first arrow above). In addition
to the molecules mentioned already from FIG. 1 and FIG. 2, the
ligand pairs also have a single-strand DNA 10, 11 which is
complementary respectively also to the single-strand DNA bonded
covalently to the ligand (otherwise the molecules and references
are identical to FIG. 1). This additional single-strand DNA 10, 11
can be generated for example via suitable primers and a PCR
reaction. The lower arrow 12 symbolises a ligation reaction (e.g.
by the addition of a ligase enzyme which leads to the two
additional single-strand DNAs 10, 11 being ligated to each other
chemically covalently. The advantage hereby is that the ligated DNA
fragment is coded respectively for a specific ligand pair via the
base sequence thereof, which pair can be hence identified easily
via DNA sequencing.
[0076] FIG. 4 describes a microfluidic device according to the
invention. Immobilised receptor, which was filled into the
container via the opening 15 with valve is situated in a container
14. The container 14 is connected on the one side and on the other
side to a pipe, through which a liquid can be guided. The pipe is
in a zone which can be heated. Heating can be effected via an IR
radiator as heating source 13. Furthermore the device has a cooling
zone 17. Optionally, this zone comprises a cooling device. In
addition, the pipe, here in the cooling region 17, has an outlet 16
with a valve out of which liquid with (free) ligands can be removed
and can be supplied for analysis. In addition, after a specific
incubation time, immobilised receptor charged with high-affinity
ligands can be removed via the opening 15.
[0077] FIG. 5 shows the result of an experiment which verifies the
effectiveness of the method according to the invention. Iminobiotin
was used as ligand which has been bonded chemically covalently
respectively to ssDNA (=im-ssDNA). The im-ssDNA was divided, in
equal parts, into a first part of im-ssDNA1 and a second part of
im-ssDNA2, im-ssDNA1 and im-ssDNA 2 having a different number of
bases which are complementary to each other according to the
experiment (e.g. 6 complementary bases in the case of "6-mer").
Im-ssDNA1 was coupled chemically covalently to the fluorescent dye
Cy5 in order to enable, on the one hand, detection and
quantification of free im-ssDNA1 or the free binary complex made of
im-ssDNA1 and im-ssDNA2 without receptor and, on the other hand,
detection of the ternary im-ssDNA1 im-ssDNA2 receptor complex. As
receptor, immobilised streptavidin was used and, as solution, an
aqueous buffer with pH 9.2 was used. The Figure shows the ratio of
the quantity of ligand-receptor complex to ligands not bonded to
the receptor ("bonded/unbonded" on the y-axis) under competitive
conditions, i.e. in the presence of iminobiotin-free ssDNA (ssDNA)
as example of a non-affine ligand (see "one-arm", "6-mer", "8-mer"
and "21-mer" on the x-axis) or non-competitive conditions, i.e.
without the presence of competitive ssDNA ("21-mer, no comp." on
the x-axis).
[0078] Apart from in the non-competitive experiment ("21-mer, no
comp." on the x-axis), the iminobiotin-free ssDNA was present in
the solution in 300 times excess. In the experiment with the title
"one-arm" (also a 6-mer), only im-ssDNA1 was present, i.e. no
im-ssDNA2, so that no binary ligand complexes were able to be
formed. The "one-arm" experiment hence shows the binding ratio for
a monomeric iminobiotin to streptavidin. In direct comparison to
the "6-mer" experiment, it becomes clear that, under the tested
competitive conditions with dimeric iminobiotin (=binary ligand
complex), the binding equilibrium is displaced clearly in the
direction of ligand-receptor complex. This effect is more clearly
pronounced by the higher stabilisation due to hybridisation of 8
base pairs in the "8-mer" experiment. If the number of
complementary bases rises further however (e.g. here 21
complementary bases in the "21-mer" experiment), then the quantity
of obtained ligand-receptor complex falls to a value which
corresponds approximately to the value of the "one-arm"
experiment.
[0079] This observation can be explained by the fact that the
Cy5-labelled iminobiotin-bonded ssDNA (im-ssDNA1) is "trapped" in
low-affinity binary complexes with iminobiotin-free ssDNA (ssDNA)
and therefore can no longer bind to complementary Cy5-free, but
iminobiotin-containing, ssDNA (im-ssDNA2). This effect is observed
therefore only in the "21-mer" experiment since here a formed
im-ssDNA1 ssDNA complex is so stable that, at room temperature
(without further energy supply), dissociation of this complex in
practice no longer takes place. In the case of a number of
complementary bases of 6 and 8 bases ("6-mer" or "8-mer"), this
effect does not however occur since here the formation of the
im-ssDNA1 ssDNA complex is not static, but is dynamic and hence, in
the case of this lower number of complementary bases, an
iminobiotin-bonded ssDNA (im-ssDNA1) which is "blocked" by ssDNA
becomes free again and can react with a further iminobiotin-bonded
ssDNA (im-ssDNA2) to form a high-affinity binary complex.
Consequently, in the case of a number of base pairs of 6 and 8, an
"accumulation" of complexes made of receptor (here streptavidin)
and high-affinity ligand pairs (here a pair of two
iminobiotin-molecules) is achieved at room temperature, as a result
of which this method is superior, with respect to sensitivity, to a
static method. The sensitivity can be increased by a number of 21
base pairs being used and being alternately heated (dissociation)
and cooled (hybridisation), i.e. energy is supplied.
[0080] FIG. 6 shows a Y-form which the total complex adopts in a
preferred embodiment of the invention. The first ligand 2 which is
bonded chemically covalently to the first single-strand DNA 8 has a
portion 18 which codes for the first ligand 2. The second ligand 3
which is bonded chemically covalently to the second single-strand
DNA 9 has a portion 19 which codes for the second ligand 3. Here,
six bases of the first single-strand DNA 8 are complementary to six
bases of the second single-strand DNA 9 and, during the method, can
form base pairs 20 (see the lines between the single-strand DNAs 8,
9). Furthermore, the first single-strand DNA 8, in this embodiment,
is hybridised in regions (here: at the 3' end) with a first
additional single-strand DNA 10. The first additional single-strand
DNA 10 is characterised in that it comprises an "overhang" of two
to twelve bases (here: five bases, located at the 3' end thereof)
which are complementary to two to twelve bases of the second
single-strand DNA 9 (here: five bases) and which, with the
complementary bases, can form base pairs 20 during the method (see
the lines between the additional first single-strand DNA 10 and the
second single-strand DNA 9).
[0081] If the second ligand 3 is hybridised via the second
single-strand DNA 9 in regions with a suitable second additional
DNA 11, then formation of the total complex is effected (here: in
Y-form) such that the first additional DNA 10 is brought together
with the second additional DNA 11 such that they can be ligated
chemically covalently via addition of a ligase enzyme. If a ligase
enzyme is added in the method, then it is achieved that ligation
products of both single-strand additional DNAs 10, 11 which code
for high-affinity ligand complexes accumulate in the course of the
method. As a result, the sensitivity of detection thereof rises.
Since the accumulation product concerns DNA, this can be amplified
even further (e.g. by PCR), as a result of which the sensitivity of
the method is increased again. In addition, sequencing of the
ligated DNA allows a rapid conclusion to be made with respect to
the two ligands 2, 3 since the ligated DNA has the portions 18, 19
which codes for both ligands 2, 3.
[0082] The invention can be characterized by the following aspects:
[0083] 1. Method for sensitive identification of high-affinity
complexes made of two ligands (2, 3, 4, 5, 6, 7) and one receptor
(1), comprising the steps [0084] a) interaction of a large number
of different complexes made of ligands (2, 3, 4, 5, 6, 7) of a
chemical library with at least one receptor (1) in a solution, the
ligands (2, 3, 4, 5, 6, 7) of the library having a single-strand
DNA (8, 9) or RNA with a base length of more than 10 bases, which
is bonded chemically covalently to the ligands (2, 3, 4, 5, 6, 7),
and more than 10 bases of the single-strand DNA (8) or RNA of a
first part of the ligands (2, 6, 7) being complementary to bases of
the single-strand DNA (9) or RNA of a second part of the ligands
(3, 4, 5), and the ligands being complexed to form ligand complexes
via hybridisation of the DNA (8, 9) or RNA; [0085] b) incubation of
the solution for a specific period of time, complexes made of
ligand complexes and the receptor (1) being produced; [0086] c)
dissociation of the ligand complexes to form free ligands; [0087]
d) re-hybridisation of the free ligands to form further ligand
complexes; [0088] e) incubation of the solution for a specific
period of time, further complexes made of the further ligand
complexes and the receptor (1) being produced; [0089] f)
identification of the resulting complexes made of ligand complexes
and the receptor (1), [0090] characterised in that incubation takes
place at a temperature at which the first part of the ligands (2,
6, 7) and the second part of the ligands (3, 4, 5) are hybridised
in the solution to form ligand complexes, [0091] or comprising the
steps [0092] i) interaction of a large number of different
complexes made of ligands (2, 3, 4, 5, 6, 7) of a chemical library
with at least one receptor (1) in a solution, the ligands (2, 3, 4,
5, 6, 7) of the library having a single-strand DNA (8, 9) or RNA
with a base length of more than 2 bases, which is bonded chemically
covalently to the ligands (2, 3, 4, 5, 6, 7), and only 2 to 10
bases of the single-strand DNA (8) or RNA of a first part of the
ligands (2, 6, 7) being complementary to bases of the single-strand
DNA (9) or RNA of a second part of the ligands (3, 4, 5), and the
ligands being complexed to form ligand complexes via hybridisation
of the DNA (8, 9) or RNA; [0093] ii) incubation of the solution for
a specific period of time, complexes made of ligand complexes and
the receptor (1) being produced; [0094] iii) identification of the
resulting complexes made of ligand complexes and the receptor (1),
[0095] characterised in that the solution is incubated at a
temperature at which, between the first part of the ligands (2, 6,
7) and the second part of the ligands (3, 4, 5), an equilibrium of
hybridisation to form ligand complexes and dissociation to form
free ligands (2, 3, 4, 5, 6, 7) arises. [0096] 2. Method according
to aspect 1, characterised in that, if more than 10 bases of the
single-strand DNA (8) or RNA of a first part of the ligands (2, 6,
7) are complementary to bases of the single-strand DNA (9) or RNA
of a second part of the ligands (3, 4, 5), the ligand complexes are
dissociated by increasing the temperature of the solution, in
particular to a temperature of 35.degree. C. to 95.degree. C.,
preferably 50.degree. C. to 95.degree. C., particularly preferably
70.degree. C. to 95.degree. C., in particular 80.degree. C. to
95.degree. C. [0097] 3. Method according to one of the preceding
aspects, characterised in that, if more than 10 bases of the
single-strand DNA (8) or RNA of a first part of the ligands (2, 6,
7) are complementary to bases of the single-strand DNA (9) or RNA
of a second part of the ligands (3, 4, 5), the dissociation of the
ligand complexes is implemented in a part of the solution which is
spatially at a spacing from the receptor, preferably such that the
receptor is not functionally impaired by the conditions which lead
to the dissociation. [0098] 4. Method according to one of the
preceding aspects, characterised in that, if more than 10 bases of
the single-strand DNA (8) or RNA of a first part of the ligands (2,
6, 7) are complementary to bases of the single-strand DNA (9) or
RNA of a second part of the ligands (3, 4, 5), the free ligands are
re-hybridised by lowering the temperature of the solution, in
particular to a temperature of 1.degree. C. to 30.degree. C.,
preferably 5.degree. C. to 28.degree. C., particularly preferably
10.degree. C. to 25.degree. C., in particular 15.degree. C. to
20.degree. C. [0099] 5. Method according to one of the preceding
aspects, characterised in that, if more than 10 bases of the
single-strand DNA (8) or RNA of a first part of the ligands (2, 6,
7) are complementary to bases of the single-strand DNA (9) or RNA
of a second part of the ligands (3, 4, 5), steps c) to e) are
repeated at least once or twice, preferably at least 3 or 4 times,
particularly preferably at least 5 or 6 times, in particular at
least 7 or 8 times. [0100] 6. Method according to one of the
preceding aspects, characterised in that, if more than 10 bases of
the single-strand DNA (8) or RNA of a first part of the ligands (2,
6, 7) are complementary to bases of the single-strand DNA (9) or
RNA of a second part of the ligands (3, 4, 5), at least 20 bases,
preferably at least 40 bases, particularly preferably at least 100
bases, in particular at least 200 bases, of the single-strand DNA
(8) or RNA of a first part of the ligands (2, 6, 7) are
complementary to the single-strand DNA (9) or RNA of a second part
of the ligands (3, 4, 5). [0101] 7. Method according to one of the
preceding aspects, characterised in that, if only 2 to 10 bases of
the single-strand DNA (8) or RNA of a first part of the ligands (2,
6, 7) are complementary to bases of the single-strand DNA (9) or
RNA of a second part of the ligands (3, 4, 5), only 3 to 9 bases,
preferably only 5 to 9 bases, particularly preferably only 6 to 8
bases, of the single-strand DNA (8) or RNA of a first part of the
ligands (2, 6, 7) are complementary to the single-strand DNA (9) or
RNA of a second part of the ligands (3, 4, 5). [0102] 8. Method
according to one of the preceding aspects, characterised in that
the length of the DNA (8) or RNA of the first part of the ligands
(2, 6, 7) and/or the length of the DNA (9) or RNA of the second
part of the ligands (3, 4, 5), is at least 20 bases, preferably at
least 40 bases, particularly preferably at least 100 bases, in
particular at least 200 bases. [0103] 9. Method according to one of
the preceding aspects, characterised in that the first part of the
ligands (2, 6, 7) has L different ligands (2, 6, 7) and the second
part of the ligands (3, 4, 5) has M different ligands (3, 4, 5) so
that LM different ligand complexes are formed. [0104] 10. Method
according to one of the preceding aspects, characterised in that
the solution is incubated in at least one of steps b), e) and ii)
at a temperature of 1.degree. C. to 50.degree. C., preferably
5.degree. C. to 37.degree. C., particularly preferably 10.degree.
C. to 25.degree. C., in particular 15.degree. C. to 20.degree. C.
[0105] 11. Method according to one of the preceding aspects,
characterised in that the solution is incubated in at least one of
steps b), e) and ii) for a period of time of 0.1 to 48 hours,
preferably 0.2 to 24 hours, particularly preferably 0.5 to 12
hours, in particular 1 to 6 hours. [0106] 12. Method according to
one of the preceding aspects, characterised in that the receptor
(1) is immobilised, preferably on a substrate selected from the
group consisting of glass, ceramic, biopolymer, sepharose,
synthetic polymer and hydrogel. [0107] 13. Method according to one
of the preceding aspects, characterised in that the receptor (1)
comprises a protein, a DNA, an RNA, a cell and/or an organic
molecule with a molecular mass 200 kDa, preferably 100 kDa,
particularly preferably 10 kDa, in particular 3 kDa, or consists
thereof. [0108] 14. Method according to one of the preceding
aspects, characterised in that the ligands (2, 3, 4, 5, 6, 7)
comprise a molecule selected from the group consisting of protein,
peptide, lipid, carbohydrate, dsDNA, ssDNA, dsRNA and ssRNA,
aptamer, organic molecule with a molecular mass 200 kDa, preferably
100 kDa, particularly preferably 10 kDa, in particular 3 kDa, or
consist thereof. [0109] 15. Method according to one of the
preceding aspects, characterised in that the complexes made of
ligand complexes and the receptor (1) are identified via an
analytical method, preferably selected from the group consisting of
mass spectrometry, HPLC, gas chromatography, IR spectroscopy and
DNA sequencing, in particular via DNA sequencing of a DNA- or RNA
barcode. [0110] 16. Method according to one of the preceding
aspects, characterised in that the single-strand DNA (8) or RNA in
the first part of the ligands (2, 6, 7) and/or the single-strand
DNA (9) or RNA in the second part of the ligands (3, 4, 5)
comprises a base sequence which codes for the chemically
covalently-bonded ligands, this base sequence being hybridised
preferably at least in regions to form a complementary base
sequence of a further single-strand DNA or RNA. [0111] 17. Method
according to one of the preceding aspects, characterised in that
the single-strand DNA (8, 9) or RNA of the ligands (2, 3, 4, 5, 6,
7) of the library is hybridised in regions to form a complementary
base sequence of a further single-strand DNA or RNA, 2 to 10 bases
of the further single-strand DNA or RNA of a first part of the
ligands (2, 6, 7) being complementary to bases of the single-strand
DNA (9) or RNA of a second part of the ligands (3, 4, 5) and
binding to the complementary bases, during the method, preferably a
Y-form being configured, and a ligase being added during the
method, which ligates chemically covalently the further
single-strand DNA or RNA of the first part of the ligands (2, 6, 7)
to the further single-strand DNA or RNA of the second part of the
ligands (3, 4, 5). [0112] 18. Method according to aspect 17,
characterised in that, in the case of a single-strand DNA (8) or
RNA of a first part of the ligands (2, 6, 7), [0113] i) which has
the ligand (2, 6, 7) at the 5' end thereof, has the bases which are
complementary to the bases of the single-strand DNA (9) or RNA of a
second part of the ligands (3, 4, 5), at the 5' end thereof, and
the 2 to 10 bases of the further single-strand DNA (10) or RNA,
which are complementary to bases of the single-strand DNA (9) or
RNA of a second part of the ligands (3, 4, 5), are disposed at the
3' end of the further single-strand DNA (10) or RNA; or [0114] ii)
which has the ligand (2, 6, 7) at the 3' end thereof, has the
bases, which are complementary to the bases of the single-strand
DNA (9) or RNA of a second part of the ligands (3, 4, 5), at the 3'
end thereof, and the 2 to 10 bases of the further single-strand DNA
(10) or RNA, which are complementary to bases of the single-strand
DNA (9) or RNA of a second part of the ligands (3, 4, 5), are
disposed at the 5' end of the further single-strand DNA (10) or
RNA. [0115] 19. Microfluidic device for implementing the method
according to one of the aspects 1 to 18, characterised in that the
device comprises: [0116] a) a container (14) for receiving
immobilised receptor (1), the container (14) having an access (15)
with valve which serves for injection of ligands (2, 3, 4, 5, 6, 7)
of a library and for isolation of ligand-receptor complexes and the
container (14) having an inlet and an outlet for a fluid pipe;
[0117] b) a fluid pipe which is connected to an inlet and an outlet
of the container (14) and which has an outlet (16) with valve at
one place; [0118] c) a heating device (13) which is disposed in a
first region of the fluid pipe; and [0119] d) a cooling region (17)
which is situated in a second region of the fluid pipe, different
from the first. [0120] 20. Microfluidic device according to aspect
19, characterised in that the cooling region (17) has a cooling
device and/or the cooling region (17) is disposed in the region of
the outlet (16) with valve. [0121] 21. Microfluidic device
according to one of the aspects 19 or 19, characterised in that the
device has a pump which is disposed such that it can pump a liquid
through the fluid pipe and/or the container (14) of the device has
filters which prevent passage of immobilised receptor into the
fluid pipe. [0122] 22. Chemical library, comprising ligands (2, 3,
4, 5, 6, 7) which have bonded chemically covalently a single-strand
DNA (8, 9) or RNA with a base length of more than 2 bases,
characterised in that only 2 to 10 bases of the single-strand DNA
(8) or RNA of a first part of the ligands (2, 6, 7) are
complementary to bases of the single-strand DNA (9) or RNA of a
second part of the ligands (3, 4, 5). [0123] 23. Library according
to aspect 22, characterised in that only 3 to 9 bases, preferably
only 5 to 9 bases, particularly preferably only 6 to 8 bases, of
the single-strand DNA (8) or RNA of a first part of the ligands (2,
6, 7) are complementary to the single-strand DNA (9) or RNA of a
second part of the ligands (3, 4, 5). [0124] 24. Library according
to one of the aspects 22 or 23, characterised in that the length of
the DNA (8) or RNA of the first part of the ligands (2, 6, 7)
and/or the length of the DNA (8) or RNA of the second part of the
ligands (3, 4, 5) is at least 20 bases, preferably at least 40
bases, particularly preferably at least 100 bases, in particular at
least 200 bases. [0125] 25. Library according to one of the aspects
22 to 24, characterised in that the first part of the ligands (2,
6, 7) has L different ligands (2, 6, 7) and the second part of the
ligands (3, 4, 5) has M different ligands (3, 4, 5) so that the
library has LM different ligand complexes. [0126] 26. Library
according to one of the aspects 22 to 25, characterised in that the
ligands (2, 3, 4, 5, 6, 7) comprise a molecule selected from the
group consisting of protein, peptide, lipid, carbohydrate, dsDNA,
ssDNA, dsRNA and ssRNA, aptamer, organic molecule with a molecular
mass 200 kDa, preferably 100 kDA, particularly preferably 10 kDa,
in particular 3 kDa, or consist thereof. [0127] 27. Library
according to one of the aspects 22 to 26, characterised in that the
single-strand DNA (8, 9) or RNA of the ligands of the library is
hybridised respectively in regions to form a complementary base
sequence of a further single-strand DNA (10, 11) or RNA, 2 to 10
bases of a first further single-strand DNA (10) or RNA of a first
part of the ligands (2, 6, 7) being complementary to bases of the
second single-strand DNA (9) or RNA of a second part of the ligands
(3, 4, 5). [0128] 28. Library according to aspect 27, characterised
in that the single-strand DNA (8) or RNA of a first part of the
ligands (2, 6, 7), [0129] i) which has the ligand (2, 6, 7) at the
5' end thereof, has the bases, which are complementary to the bases
of the single-strand DNA (
9) or RNA of a second part of the ligands (3, 4, 5), at the 5' end
thereof, and the 2 to 10 bases of the further single-strand DNA
(10) or RNA, which are complementary to bases of the second
single-strand DNA (9) or RNA of a second part of the ligands (3, 4,
5), are disposed at the 3' end of the further single-strand DNA
(10) or RNA; or [0130] ii) which has the ligand (2, 6, 7) at the 3'
end thereof, has the bases, which are complementary to the bases of
the single-strand DNA (9) or RNA of a second part of the ligands
(3, 4, 5), at the 3' end thereof, and the 2 to 10 bases of the
further single-strand DNA (10) or RNA, which are complementary to
bases of the single-strand DNA (9) or RNA of a second part of the
ligands (3, 4, 5), are disposed at the 5' end of the further
single-strand DNA (10) or RNA. [0131] 29. Use of the library
according to one of the aspects 22 to 28 for selective and
sensitive identification of high-affinity ternary ligand-receptor
complexes.
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