Modified nucleotides for synthesis of nucleic acids, a
kit containing such nucleotides and their use for the
production of synthetic nucleic acid sequences or genes
5 The present invention falls within the field of the
10
15
20
synthesis of functionalized copolymers of biological
interest. It relates more particularly to nucleotides
required for the synthesis of nucleic acids, in
particular of very long nucleic acids, to a kit
containing such nucleotides and to the use thereof for
the production of synthetic nucleic acid sequences or
genes.
Prior art
To date, there are two major categories of in vitro
nucleic acid synthesis: chemical syntheses and
enzymatic syntheses.
The most common in vitro nucleic acid chemical
synthesis method is the method of polymerization by
means of phosphoramidi tes described by Adams et al.
(1983, J. Amer. Chern. Soc. 105:661) and Froehler et a1.
(1983, Tetrahedron Lett. 24:3171). In this method, each
25 nucleotide to be added is protected on the 5'-0H group
so as to prevent uncontrolled polymerization of several
nucleotides of the same type. Generally, the protection
of the 5'-0H group is carried out with a trityl group.
In order to prevent any degradation due to the use of
I 30 powerful reagents, the bases borne by thE; nucleotides
can also be protected. Generally, the protection used
involves an
Nucleosides
isobutyryl group
& Nucleotides
(Reddy
16:1589).
et al. 1997'
After each
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2
incorporation of new nucleotides, the 5' -OH group of
the last nucleotide of the chain undergoes a
deprotection reaction for the purpose of making it
available for the subsequent polymerization step. The
5 nitrogenous bases borne by the nucleotides which make
up the nucleic acid, themselves, are deprotected only
after the complete polymerization has been finished.
These chemical synthesis methods proved to be expensive
10 and dangerous to use owing to the nature of the
reagents involved. Furthermore, they are inefficient
for the synthesis of long nucleic acid fragments
(fragments greater than about a hundred nucleotides) .
15 With a view to developing methods of nucleic acid
synthesis, in particular for the production of very
long fragments with high yields, and also methods
compatible with the already existing genetic elements,
such as DNA plasmids, synthesis techniques have been
20 developed which use enzymatic catalysts for carrying
out the coupling reaction between the nucleotides, even
in the absence of a template strand.
In some of these enzymatic synthesis methods, the
25 enzyme enabling the polymerization is directly added to
the natural nucleotides (Deng et al. 1983, Meth.
Enzymol. 100:96). Starting from an initial nucleic acid
fragment known as a primer, the polymerization enzyme
and also nucleotides of one and the same type are
30 added. The polymerization reaction is then initiated,
and the nucleic acid grows sequentially by repetition
of these steps of creating a phosphodiester bond, until
wo 2016/034807
said polymerization is
chemical method.
PCT/FR2015/052310
3
stopped by a physical or
The use of natural nucleotides (that is to say
5 unmodified and unprotected nucleotides) leads to an
uncontrolled polymerization resulting in a very
heterogeneous mixture of nucleic acid molecules. This
is because nothing prevents the addition of several
nucleotides of one and the same type after a first
10 addition. In practice, such a synthesis method proves
to be unusable for the synthesis of nucleic acid
fragments having a desired sequence.
The use of protected nucleotides makes it possible, to
15 a certain extent, to solve this uncontrolled
polymerization phenomenon. The protected nucleotides
make it possible to stop the synthesis by totally or
partially preventing the creation of phosphodiester
bonds subsequent to that desired.
20
Nucleotides are the "monomers" used for nucleic acid
synthesis. Their chemical properties and also their
ability to react or not react guarantee that the
desired synthesis takes place correctly. It is
25 important to be able to polymerize the nucleotides one
by one in the desired order in order to be able to
synthesize a nucleic acid fragment comprising the
desired sequence. This polymerization is equivalent to
the addition of nucleotides one after another in an
30 order which must be strictly adhered to. It is in
particular necessary to take care that several
nucleotides comprising tbe same nitrogenous base and
introduced at the same time do not react in chain,
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4
leading to the uncontrolled growth
chain and, as a result, to the
erroneous nucleic acid sequence.
PCT/FR2015/052310
of the oligomeric
production of an
5 There are modified nucleotides comprising certain
structural modifications compared with natural
nucleotides, which give them certain advantages when
they are used for nucleic acid synthesis. They are
generally obtained by chemical or enzymatic
10 modifications of the nucleotides naturally present in
cells. Some modified nucleotides are said to be
protected because they comprise chemical groups which
prohibit the modification of a chemical function to be
preserved during other reactions. The protective groups
15 may be placed at various sites of the nucleotide
molecule.
One particular class of protected nucleotides has a
polymerization-reaction-terminating function. The role
20 of these "chain-terminating" nucleotides consists in
preventing excessive and undesired polymerization of
the nucleotides introduced into the reaction medium.
When a terminator nucleotide is incorporated into a
nucleic acid molecule, it impedes the subsequent
25 polymerization of another nucleotide. Thus, a single
nucleotide can be added to each nucleic acid molecule
during the elongation step. Even if the various
nucleotides, making up the nucleic acid fragment to be
synthesized, are introduced sequentially, it is
30 necessary to use "terminator'~ nucleotides in order to
prevent undesirable repetition phenomena.
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5
The use of "terminator" nucleotides guarantees the
reliability and reproducibility of nucleic acid
synthesis methods, whether they are chemical or
enzymatic. They can have a great influence on the
5 synthesis performance levels of a given method.
The protected nucleotides used for the chemical
synthesis of nucleic acids comprise, in the 5'-0H
position, a protection by covalent bonding to a DMT
10 (4,4'-dimethoxytrityl) group and, in the 3'-0H
position, a phosphoramidite group acting as a catalyst
for the polymerization reaction of the .nucleotides with
one another. These nucleotides comprising DMT and
phosphoramidite groups are called protected
15 phosphoramidite nucleotides.
uncontrolled polymerization
group protecting the 5'-0H.
The protection
is provided by
During chemical
against
the DMT
nucleic
acid synthesis, a first deprotection phase, called
detri tylation, takes place in order to remove the DMT
20 group and to obtain a 5'-0H group that is available to
react with the nucleotide to be inserted. It is
particularly important to have the most efficient
deprotection reaction possible in order to allow the
addition of the next nucleotide in all cases.
25
Protected phosphoramidite nucleotides are exclusively
used during the chemical synthesis of nucleic acids.
Their "terminator" function is in fact provided by the
DMT group bonded to the 5'-0H. Since the chemical
30 synthesis takes place in' the 3' to 5' direction, the
existence of a DMT group protecting the 5'-0H makes it
possible to prevent any excessive polymerization until
the next deprotection step.
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6
Thus, protected phosphoramidite nucleotides are not
suitable for enzymatic synthesis methods.
5 Some "terminator" nucleotides have also been developed
for "second-generation" sequencing methods. However, in
addition to being totally unsuitable for nucleic acid
synthesis, the terminator nucleotides used for the
sequencing have a certain number of totally
10 unacceptable limitations.
The main limitation is their ability to be used by the
elongation enzymes. This is because the fluorescent
labels bonded to the terminator nucleotides for the
15 sequencing have a considerable size. However, the
elongation enzymes have an extremely small amount of
space in their active site and thus have little chance
of being able to accept, in order to polymerize them,
terminator nucleotides housing imposing fluorescent
2 0 groups, such as groups comprising conjugated aromatic
rings.
Modern DNA sequencing techniques are based on
complementary interactions between a template strand,
25 the sequenced strand, and a strand undergoing
elongation. Generally, the modified nucleotides used
for the sequencing must have intact properties of
pairing with their complementary nucleotides. The
modified nucleotides should preserve these properties
30 of interactions that are essential for their use.
However, the nitrogenous bases which constitute the
modified nucleotides for sequencing are analogs of
natural nitrogenous bases such as adenine, guanine,
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7
thymine, uracil and cytosine, and thus do not have the
same chemical structure: some atoms are substituted
with others and some groups are added or deleted. These
unnatural nitrogenous bases can have many drawbacks,
5 such as that, for example, of not being recognized by
living organisms.
Once incorporated, the terminator nucleotides are
deprotected in order to allow the addition of the next
10 nucleotide. The deprotection step involves a physical
or chemical means which makes it possible to delete the
group· responsible for the terminator function. The
other functional groups associated with the modified
nucleotide are deleted during similar deprotection
15 steps. Several deprotection steps are thus generally
necessary during the various sequencing processes in
order to be able to move to the determination of the
next nucleotide. These various deprotection steps
accumulate and multiply the use of powerful reagents or
20 of extreme physical conditions which promote the
degradation of the various species present in the
reaction medium and in particular the degradation of
the nucleic acids. Furthermore, a large number of
deprotection steps considerably decreases the rapidity
25 of the process and its performance levels.
Yet another major problem encountered during the use of
modified nucleotides for sequencing is the appearance
of scars after the deprotection steps. The various
30 chemical structures which serve as a link between the
functional groups and the nucleotide are capable of
being broken during the deprotection steps. However,
this breaking does not make it possible to separate all
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8
of the chemical bonding structures. Thus, more or less
large parts of these structures remain attached to the
nucleotides, despite the various deprotection steps.
These residues have a very harmful effect on the
5 sequencing process and on any use or modification of
the nucleic acids in general.
Whatever the structure of the nucleotide retained, the
existing modified nucleotides do not make it possible
10 to meet the expectations of enzymatic synthesis
methods. Their mediocre use by elongation enzymes, the
positioning of the various functional groups, the
systematic
obligation
use of modified nitrogenous bases,
of preserving the interactions with
the
the
15 complementary nucleotides, the numerous deprotection
steps and the presence of residual scars prohibit the
use of these nucleotides for enzymatic nucleic acid
synthesis.
20 To date, there is therefore no satisfactory technical
solution proposing protected nucleotides that are
compatible with the enzymatic synthesis of nucleic
acids, in particular for the enzymatic synthesis of
very long nucleic acid fragments.
25
Aims of the invention
A first aim of the invention is to provide modified
nucleotides which are sui table for enzymatic nucleic
30 acid synthesis.
Another aim of the invention is to provide natural
nucleotides which are modified by various functional
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9
groups making it possible to render them compatible
with their use during a nucleic acid synthesis process.
Another aim of the invention is to provide modified
5 nucleotides which make it possible to synthesize very
long nucleic acids, that is to say nucleic acids of at
least several hundred or several thousand nucleotides,
and more particularly according to the process
described in the patent application by the same
10 applicant, not yet published, FR 14-53455.
15
Description of the invention
To this effect, the present invention provides a
modified nucleotide, intended for the enzymatic
synthesis of nucleic acids, such as long-chain nucleic
acids, comprising a "natural" nitrogenous base or a
natural nitrogenous base analog, a ribose or
20 deoxyribose carbohydrate, and at least one phosphate
group, characterized in that it comprises at least one
R or R' group, called modifier group, borne:
- by said natural nitrogenous base or analog,
- and/or by the oxygen in position 3' of the ribose or
25 deoxyribose molecule, making it possible to block the
polymerization of said nucleotide (the modifier group
is then a protective group) and/or to enable the
interaction of said nucleotide with another molecule,
30
different than another nucleotide,
during nucleic acid synthesis, R
one functional end group (which
effector group) .
such as a protein,
comprising at least
may also be called
5
10
15
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10
More particularly, the modifier group is advantageously
not a large group, such as a group comprising
conjugated aromatic rings, in particular so as to allow
access of the enzyme to the reaction site.
The nucleotide according to the invention may be a
monophosphate, a diphosphate or a triphosphate, the
phosphate
unmodified.
group(s) being free, that is to say
The nucleotide according to the invention is in the
form of one of formulae (I), (III) and (IV) below:
(t} R
.,.,o~ I 1 /~.__> . __..Baso
(PPJPO
3 /."
I ( OH)
z
R' l (M)
!
~ (formula I)
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11
(formula III)
HO 0 I T (formula IV)
5 in which:
(PP)PO represents a mono-, di- or triphosphate group,
(OH) describes the possibility of a ribose or
deoxyribose molecule,
T is a hydrogen, or a cleavable radical chosen from
10 -NHz, -N3, -(C=O)H, -CnHzn+l with n between 1 and 30,
preferably between 1 and 12, -trimethylsilyl,
-phosphate, -S03, - (C=O) OCnHzn+l with n between 1 and 30,
preferably between 1 and 12, -(C=O)SCnHzn+l with n
'between 1 and 30, preferably between 1 and 12,
15 -nitrobenzene, -benzyl, -halobenzyl, -amide,
-carbonate, -benzoyl, -peroxyl, -nitrile, -thiol,
wo 2016/034807
-imide, -carbamate,
-halophenyl, -picolyl,
12
-cyanate,
PCT/FR2015/052310
-alkyne, -phenyl,
M, which is optionally present, lS a group covalently
bonded to Q and to Z, M being chosen from alkyl,
5 alkenyl, alkyne, aryl, alkylaryl, heteroaryl, acyl,
alkyloxy, alkylamino, alkoxyamino, amido, alkylimido,
alkenylimido, arylimido, fluoroalkyl, alkylphosphate,
alkyl thio, thioacyl, alkyl sulfonyl, aryl sulfonyl,
alkylsulfinyl, alkylammonium, alkylsulfonium,
10 alkylsilyl, alkylcarbonyl, alkylcarbanyl,
alkylcarbamoyl or alkylhydroxylamino,
Z is a cleavable group, chosen from -0-, -S-, =SH-,
=S=, =S-, -SiH2-, =SiH-, =Si=, =Si-, -Se-, =SeH-, =Se-,
=Se=, -SeH2-, -PH-, =P-, =PH=, =P=, =PH-, -PH3-, -AsH-,
15 =As-, =AsH=, =As=, =AsH-, -ASH3-, amine, ester, silyl,
alkyl, benzyl, nitrobenzyl, amide, carbonate, benzoyl,
peroxyl, nitrile, thiol, imide, carbamate, cyanate,
hydroxylamine, sulfoxide, sulfonate, thiosulfinate,
thioester, acyl halide, hypoiodyl, alkyne, halophenyl,
20 halobenzyl, picoyl, diol or disulfide, or chosen from
-CH2 or -NH- when M is a -nitrobenzyl-, a -nitrotolyl-,
a -nitroxylyl-, a -nitronaphthyl- or a -nitrophenyl-,
Q is an end functional, or effector, group of the R or
R' group, Q being chosen from biotin, a protein, a
25 polynucleotide of defined sequence, a carbohydrate, an
antigen, a hormone, a neurotransmitter, a glycoside
such as digoxin, a sulfur-containing radical, in
particular bearing a thiol function, such as
glutathione, or a bidentate ligand such as catechol,
30 R and R' possibly being present independently or
simultaneously, and when R and R' are present
simultaneously:
the Z groups may be identical or different,
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13
the M groups may be identical or different,
the Q groups may be identical or different,
"base" represents a "natural" nitrogenous base chosen
from adenine, thymine, cytosine, guanine or uracil or a
5 natural nitrogenous base analog, with the exception of
thymine when R' is present and Q comprises biotin.
Preferably, T, when it is not hydrogen, and also R and
R', constitute groups which provide the chain
10 termination of the elongation step of a nucleic acid
synthesis process.
The term "cleavable radical
mean a radical or a group
or group"
covalently
is intended to
bonded to the
15 oxygen in position 3' or 2' of the ribose molecule or
in position 3' of the deoxyribose molecule, or to an
atom of the nitrogenous base, it being possible for
said bond to be broken chemically or photochemically.
Advantageously, the breaking of all of the bonds of the
20 cleavable radicals or groups T and Z of the nucleotide
molecule according to the invention is carried out
entirely and simultaneously, that is to say during the
same "deprotection" step, in particular by application
of one and the same condition or by the combined action
25 of one and the same reagent.
This deletion of the modifier groups is preferably
total in order to result in the generation of a
nucleotide free of any modifier group, that is to say
30 identical to a natural nucleotide (except for the
structure of the nitrogenous base) .
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14
As previously indicated, the R and R' groups are groups
which advantageously provide the chain ending of the
elongation step of a nucleic acid synthesis process.
These R and R' groups can have properties, by means of
5 the functional group Q placed at the free end of R and
R', of attachment to another molecule, different than a
nucleic acid, for example a molecule present on a
support.
10 This is because, during the nucleic acid synthesis
process, for example in the process described in
FR 14-53455 mentioned above, some steps assume that the
modified nucleotides used can interact with solid
supports. These solid supports comprise, at their
15 surface, molecules, proteins or chemical functions that
are compatible with the modifier groups of the present
nucleotides. This functionality of the modified
nucleotides is then essential for the nucleic acid
synthesis process to take place correctly. In one
20 preferred embodiment, the modified nucleotides comprise
a group which allows them to become combined with a
solid support in order to be purified, for example, by
forming, with the molecules present at the surface of a
solid support, combination complexes having a very low
25 dissociation constant, in particular less than
10-6 mol/1. Following the deprotection step, the group
providing this combination function is then capable of
being destroyed, thus eliminating the interaction
between the nucleotide and the solid support. In this
30 case, the nucleotide according to the invention has a
double advantage, namely the presence of a group
allowing purification and the capacity for destruction
WO -2016/034807 PCT/FR2015/052310
15
of this same group simultaneously with the other
modifier groups, on one and the same nucleotide.
Preferably, the modifier group R is borne by the
5 nitrogenous base and forms one of the structures (V)
below:
HN-Z-M-Q
0
Adenine-based structure (Va) Thymine-based structure (Vt)
- 7 '(N
9N
0
/
Sugar
1
4 -~N-Z~-,-2 ;'1/1-Q
HN-Z2
10 Cytosine-based structure (V0 ) Guanine-based structure (Vg)
Uracil-based structure (Vu)
in which structures:
"sugar" represents the bond between said nitrogenous
base and the ribose or deoxyribose molecule of the
15 nucleotide molecule,
Z1 and Z2 are identical or different cleavable Z groups.
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16
The Z, M and Q groups have the meanings previously
described.
5 In this embodiment of the invention, the nucleotides
are modified with groups borne by the atoms of the
nitrogenous bases usually involved in Watson-Crick
pairing mechanisms, that is to say borne by the
nitrogen atoms of the amine functions normally involved
10 in the pairing with a complementary nucleotide. The
attachment of the various modifier groups to the atoms
consisting the nitrogenous bases is always carried out
by means of the Z groups, of cleavable type, via a bond
of covalent type.
15
20
25
30
In the case of a nitrogenous base of adenine type, one
preferred embodiment of the present invention consists
in bonding the modifier group to the primary amine
group 6-NH2 (structure Va).
In the case of a nitrogenous base of thymine type,
another preferred embodiment of the present invention
consists in bonding the modifier group to the secondary
amine group 3-NH (structure Vt).
In the case of a nitrogenous base of cytosine type,
another preferred embodiment of the present invention
consists in bonding the modifier group to the primary
amine group 4-NH2 (structure Vc).
In the case of a nitrogenous base of uracil type,
another preferred embodiment of the invention consists
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17
in bonding the modifier group to the secondary amine
group 3-NH (structure Vu).
In the case of a nitrogenous base of guanine type,
5 another preferred embodiment of the present invention
consists in bonding the modifier group to one of the
two, or simultaneously to both, amine groups, one being
secondary 1-NH and the other primary 2-NH2 , by means of
cleavable groups (structure Vg). In the particular case
10 where the two amine groups 1-NH and 2-NH2 are
simultaneously used to bear the modifier groups, a
ring, preferentially composed of 6 atoms, may occur
between the various separating subgroups. This ring
optionally brings about stabilization of the structure
15 of the modifier groups.
20
In these embodiments where the modifier group is borne
by the nitrogen base, the 3'0H and/or 2'0H sites of the
nucleotides are free, promoting their use as substrates
for the elongation enzymes during nucleic acid
synthesis.
Intermolecular hydrogen bonds can occur. Indeed, the
modifier group R borne by the nitrogenous base can form
25 one of the structures (VI) below:
00----H-X'/ M-0
I
e /~--<:::..3
---z
Sugar o
Adenine-based structure (VIa) Thymine-based structure (VIt>
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18
Cytosine-based structure (VI0 ) Guanine-based structure (VI9)
0----H-X1
// \ /!
5 r;---!,.1 M -Q II 4\ . 6\ ,N3 / N-\ Sugar/" 1
0 Uracil-based structure (VIu)
in which:
5 "Sugar" represents the bond between said nitrogenous
base and the ribose or deoxyribose molecule of the
nucleotide molecule,
which may be identical or different,
represent nitrogen, oxygen or sulfur atoms borne by M
10 and capable of forming, with said nitrogenous bases of
the modified nucleotide, intramolecular hydrogen bonds
(these hydrogen
intermolecular
conventional
15 nucleotides) .
bonds
hydrogen
pairings
are then similar to the
bonds observed during
between complementary
This configuration reinforces the stability of the
modified nucleotides. It also influences the
compactness of the modifier groups and allows their use
20 by elongation enzymes that are usually dependent on the
presence of a template strand.
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19
In the case of a nitrogenous base of adenine type, one
preferred embodiment of the present invention consists
in bonding the modifier group to the primary amine
5 group 6-NH2 (structure VIa) . The X1 group present
promotes the creation of an intramolecular hydrogen
bond with the nitrogen atom 1 of the purine ring. Thus,
the presence of a complementary thymine is mimicked.
10 In the case of a nitrogenous base of thymine type, one
preferred embodiment of the present invention consists
in bonding the modifier group to. the secondary amine
group 3-NH (structure VIt). The X1 group present
promotes the creation of an intramolecular hydrogen
15 bond with the oxygen atom in position 4 of the
pyrimidine ring. Thus, the presence of a complementary
adenine is mimicked.
In the case of a nitrogenous base of cytosine type, one
20 preferred embodiment of the present invention consists
in bonding the modifier group to the primary amine
group 4-NH2 (structure VIc). The X1 and X2 groups present
promote the creation of intramolecular hydrogen bonds
with the nitrogen atom in position 3 and the oxygen
25 atom in position 2 of the pyrimidine ring. Thus, the
presence of a complementary guanine is mimicked.
In the case of a nitrogenous base of guanine type, one
preferred embodiment of the present invention consists
30 in bonding the modifier group to the two amine groups,
the first being secondary 1-NH and the second primary
2-NH2 (structure VIg), by means of cleavable Z1 and Z2
groups. The X1 group present promotes the creation of
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20
the intramolecular hydrogen bond with the oxygen atom
in position 6 of the purine ring. Thus, the presence of
a complementary cytosine is mimicked.
5 In the case of a nitrogenous base of uracil type, one
preferred embodiment of the invention consists in
bonding the modifier group to the secondary amine group
3-NH (structure VIu). The X1 group present promotes the
creation of a hydrogen bond with the oxygen in position
10 4 of the pyrimidine ring. Thus, the presence of a
complementary adenine is mimicked.
It is not necessarily the mission of the modified
nucleotides according to the present invention to pair
15 with a possible complementary nucleotide borne by any
template strand. During the use of the modified
nucleotides which are subjects of the present
invention, for the generation of nucleic acids, said
nucleotides are not necessarily intended to be
20 incorporated by complementary interaction with a
possible template strand.
The modified nucleotides which are subjects of the
present invention have characteristics which allow them
25 to lose their modifier groups during specific steps.
30
After the loss of all their modifier groups, the
nucleotides of the present invention thus transformed
then recover their ability to pair with complementary
nucleotides borne by template strands.
According to one particular embodiment, the nucleotide
according to the invention can then be used as a
substrate for polymerases that are normally dependent
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21
on the presence of a template nucleic acid strand
complementary to the strand undergoing synthesis, even
in the absence of a complementary strand.
5 The functional end radical Q of the R or R' group is
preferably capable of allowing, during nucleic acid
synthesis, the attachment of said nucleotide to a solid
support, by means of a molecule other than a nucleic
acid, such as a protein, attached to the surface of
10 said support, and more particularly capable of
interacting with molecules other than a nucleic acid,
according to one or another of the following
interaction pairs: antigen/antibody, hormone/receptor,
biotin/(strept)avidin, neurotransmitter/receptor,
15 polymerase/promoter, digoxin/antidigoxin,
20
carbohydrate/lectin, sulfur-containing radical/metal
such as gold, glutathione/glutathione S-transferase, or
else bidentate ligand/metal oxide.
The metal oxides may be,
Fe304, Ga203, In203, Cr203,
Mn203, V 203, Mo02 .
for example,
Al203, ZnO,
Ti02, Zr02,
CuO, Cu203,
The bidentate ligands may be catechol, hydroxamate or a
25 hydroxycarboxylate.
The T radical is termed "blocker" in that it protects
the 3' hydroxyl group or the 2' hydroxyl group of the
carbohydrate
30 addition.
Preferably, T
nucleic acid
against any
and Z, or Z1,
synthesis,
additional nucleotide
are cleavable,
irradiation of
during
said
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22
nucleotide by means of electromagnetic radiation having
a wavelength of between 10-3 and 10-11 meters, in
particular by exposure to ultraviolet radiation.
5 Advantageous embodiments of the invention relate to the
following nucleotides:
- a nucleotide for which X1 and X2 are -NH, T is -NH2 , Z
is -CH2 , M is methylnitrobenzyl-, and Q is -biotin,
- a nucleotide for which X1 and X2 are -NH, T is -NH2 ,
10 Z, Z1 and Z2 are each -0-, M is -nitronaphthyl- and Q is
-biotin,
- a nucleotide of formula (I) bearing only the R' group
in which: Z is - (C=O) -, M is -C8H16- and Q is -NHbiotinyl,
15 - a nucleotide of particular structure (V) in which Z,
Z1 and Z2 are each -(COO)-, M is -tert-butylnitrobenzyland
Q is -NH-biotinyl.
The present invention also relates to the use of
20 nucleotides, as described previously, in a process for
the production of genes, of synthetic nucleic acid
sequences, of DNA, of RNA or of nucleic acid polymers,
in particular according to an enzymatic synthesis
process.
25
An advantageous use is for the incorporation of said
nucleotide into a polynucleotide chain previously
immobilized on a solid support, more particularly when
the polynucleotide chain is attached via its 5' end and
30 the incorporation of said nucleotide is carried out via
the 3' end of the polynucleotide chain.
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23
Nevertheless, the nucleotides according to the
invention are in free form, in combination with a
counterion if required. Although they have the capacity
to attach to a solid support, these nucleotides are
5 free from any support at the time of their
incorporation into the polynucleotide chain. Their
chemical structure is thus not linked to any solid
support. The free nature of the modified nucleotides is
particularly important for their use in the process
10 described in the patent application, not yet published,
FR 14-53455 since the nucleotides are added to
fragments of nucleic acids which are themselves
immobilized. These fragments are released and then, by
virtue of the effector group of the nucleotides that
15 have just been incorporated, are subsequently attached
via the opposite end to a second solid support. These
are thus complete nucleic acid chains, or
polynucleotides, of desired sequence which are attached
to a solid support, and not the nucleotides according
20 to the invention, used to construct the
polynucleotides.
The present invention also relates to a kit comprising
at least one modified nucleotide according to the
25 invention, more particularly said kit may comprise
various modified nucleotides, an elongation enzyme and
a solid support capable of attaching at least one of
said nucleotides.
30 The present invention will be described ln greater
detail by means of the illustrative examples below in
relation to the appended figures in which:
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24
figure 1 shows the various general structures of the
modified nucleotides which are subjects of the present
invention;
figure 2 presents the particular structures of the
5 modified nucleotides of formula (V);
figure 3 presents the formulae of the natural
nitrogenous bases adenine, thymine, cytosine and
guanine;
figure 4 shows examples of modifier groups capable of
10 interactions of intramolecular hydrogen bond type, of
formula (VI);
figure 5 represents diagrammatically the synthesis of
the compound NH2-dTTP-NitroB-Biot;
figure 6 represents diagrammatically the synthesis of
15 the compound NHrdGTP-Ni troN-Biot;
figure 7 represents diagrammatically the synthesis of
the compounds FA-Biot-dNTP;
figure 8 represents diagrammatically the synthesis of
the compound dATP-NitroB-Biot;
20 figure 9 represents diagrammatically the synthesis of
the compound dCTP-NitroB-Biot;
figure 10 shows an example of deprotection of the
polymerized compound NH2-dT-NitroB-Biot;
figure 11 shows an example of deprotection of the
25 polymerized compound NH2-dG-NitroB-Biot;
figure 12 shows an example of deprotection of the
polymerized compound FA-Biot-dNTP;
figure 13 shows an example of deprotection by
photocleavage of the polymerized compound dA-NitroB-
30 Biot;
figure 14 shows an example of deprotection by chemical
cleavage of the polymerized compound dC-NitroB-Biot;
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25
figure 15 presents an example of a modified nucleotide
according to the present invention, the Q group being a
catechol;
figure 16 represents diagrammatically the synthesis of
5 the compound FA-Cat-dNTP.
Examples
Synthesis of modified (protected) nucleotides
10 illustrative examples 1 to 6
Example 1 - Synthesis of the compound NH2-dTTP-NitroBBiot
(fig 5)
15 Step Al: 2. 2 ml of Et3N (triethylamine) and 17 5 mg of
DMAP (4-dimethylaminopyridine) and then 5.25 g of DMTCl
( 4, 4' -dimethoxytri tyl chloride) are added, at ambient
temperature overnight, to 5 g of 2'-deoxythymidine
dissolved in pyridine. 2. 4 ml of Et3N and 1. 27 ml of
20 MsCl (methanesulfonyl chloride) are then added to the
mixture. After
temperature, the
ethyl acetate.
incubation for 2 h at ambient
mixture is filtered and washed with
The filtrate is concentrated and
dissolved in 75 ml of ethanol, to which 1M of NaOH is
25 added. After refluxing for 1.5 h, the mixture is cooled
to ambient temperature and 1M of HCl is added. The
ethanol is evaporated off in a rotary evaporator and
the residue is extracted with CH2Cl2 • After silica gel
column purification, the product dTTP-Al is obtained.
30
Step A2: 1. 7 5 ml of N, N' -diisopropyl azodicarboxylate
are added, at 0°C, to a solution of 2.237 mmol of
product dTTP-Al, 2.1 g of triphenylphosphine and 1.3 g
wo 2016/034807 PCT/FR2015/052310
26
of N-hydroxyphthalimide in 50 ml of tetrahydrofuran.
After reheating at ambient temperature overnight, the
reaction product is treated with 0. 3 ml of water and
the solvent is evaporated off under vacuum. Most of the
5 impurities are eliminated by chromatography, then
giving the product dTT-A2.
Step A3: 10 equivalents of LiH in DMF are added, at
ambient temperature, to one equivalent of compound
10 dTT-A2. The mixture reacts for 30 min. The reaction is
continued by adding 3-amino-4-(bromomethyl)-5-
nitrophenylbiotirr, and then the mixture is stirred for
several hours. The product dTT-A3 is obtained.
15 Step A4: The compound dTTP-A3 is resuspended in
methanol and treated
hydrochloric acid. The
with aqueous concentrated
solution is cooled to -20°C
overnight, resulting in the product dTTP-A4.
20 Step A5: A solution of 130 mg of 2-chloro-4H-1, 2, 3-
benzodioxaphosphorin-4-one in 1.3 ml of dioxane is
added to 425 mg of 5'-0H nucleoside analog dTTP-A4
dissolved in 2 ml of pyridine and 1. 7 ml of dioxane.
The mixture is left at ambient temperature for 20 min.
25 A mixture of 1.4 rnrnol of tributylarnrnonium pyrophosphate
in DMF and 3. 2 rnrnol of tributylamine is added. After
20 min, a solution of 180 mg of iodine and 0.28 ml of
water in 14 ml of pyridine is added. After 30 min, the
reaction is stopped by adding an aqueous 5% Na 2S03
30 solution. The solvents are evaporated off under vacuum.
25 ml of water and 20 ml of CH3CN are added. The mixture
is filtered and purified by reverse-phase HPLC to give
a triphosphate compound, in the case in point dTTP-A5.
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Step A6: 0. 385 ml of cold methylhydrazine is added to
3. 725 mmol of compound dTTP-A5 in anhydrous CH3Cl2 at
-5 °C. After 10 min, a precipitate of 1, 2-dihydro-4-
5 hydroxy-2-methyl-1-oxophthalizine is formed. The
mixture is stirred for 1 h at ambient temperature. The
precipitate is removed by filtration and washed with
CH2Cl2 • The filtrate is then concentrated in a rotary
evaporator and purified by chromatography to give the
10 product NHrdTTP-Ni troB-Biot.
Example 2 - Synthesis of the compound NHrdGTP-NitroNBiot
(fig 6)
15 Step B1: 2. 4 mmol of tert-butyldimethylsilyl chloride
are added to a stirred solution of 1. 8 4 5 mmol of 2'deoxyguanine
and 326 mg of imidazole in anhydrous DMF.
The reaction is incubated with stirring at ambient
temperature for 20 h. The solvents are removed under
20 vacuum and the residue is purified by chromatography to
give the product dGTP-B1.
Step B2: 1. 7 5 ml of N, N' -diisopropyl azodicarboxylate
are added, at 0 °C, to a solution of 2. 237 mmol of
25 product dGTP-Bl, 2.1 g of triphenylphosphine and 1.3 g
of N-hydroxyphthalimide in 50 ml of tetrahydrofuran.
After reheating at ambient temperature overnight, the
reaction product is treated with 0. 3 ml of water and
the solvent is evaporated off under vacuum. Most of the
30 impurities are removed by chromatography,: then giving
the product dGTP-B2.
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28
Step B3: 3.785 mmol of compound dGTP-B2 are dried
several times using 10 ml of pyridine and evaporation
under vacuum. The residue is dissolved in 12. 5 ml of
CH2Cl2 • 9 mmol of diisopropylethylamine and 7.57 mmol of
5 6-amino-4,5-bis(iodooxy)-3-nitronaphthalen-1-yl-5-
biotin are added. When the reaction is complete, the
mixture is diluted in 100 ml of CH2Cl2 , and the organic
phase is washed with 50 ml of sodium bicarbonate and
50 ml of water. It is then dried over sodium sulfate.
10 The solvents are evaporated off under vacuum and the
prdduct is purified by chromatography to give dGTP-B3.
Step B4: 3.75 mmol of compound dGTP-B3 are dissolved in
20 ml of THF and treated with 1M of TBAF (tetra-n-
15 butylammonium fluoride) in THF. The reaction is
complete after approximately 2 h with stirring. The
mixture is extracted with CH2Cl2 and purified by
chromatography to give dGTP-B4.
20 Step B5: 425 mg of 5' -OH nucleoside analog are treated
similarly to step A5 of example 1. The final mixture is
filtered and purified by reverse-phase HPLC to give a
triphosphate compound, in the case in point dGTP-B5.
25 Step B6: 0. 385 ml of cold methylhydrazine is added to
3. 725 mmol of compound dGTP-B5 in anhydrous CH2Cl2 at
-5°C. After 10 min, a precipitate of 1,2-dihydro-4-
hydroxy-2-methyl-1-oxophthalizine is formed. The
mixture is stirred for 1 h at ambient temperature. The
30 precipitate is removed by filtration and washed with
CH2Cl2 • The filtrate is then concentrated in a rotary
evaporator and purified by chromatography to give the
product NH2-dGTP-NitroN-Biot.
wo 2016/034807
Example 3
(fig 7)
PCT/FR2015/052310
29
Synthesis of the compounds FA-Biot-dNTP
5 Step C1: 100 pl of 1M w1-biotin nonanoic acid in DMF
are mixed with 100 pl of 1M carbonyldiimidazole in DMF.
The formation of imidazolide takes place in 30 s at
ambient temperature. 100 pl of 50 roM dioxyribonucleotide
5' -triphosphate in water are then added to
10 the mixture. The product is formed in 12 h at ambient
temperature. It is then precipitated with acetone and
dissolved in water so as to be finally purified by
chromatography to give the product FA-Biot-dNTP.
15 Example 4 - Synthesis of the compound dATP-NitroB-Biot
(fig 8)
20
Step D1: 2.2 ml of Et3N and 175 mg of DMAP and then
5.25 g of DMTCl are added, at ambient temperature
overnight, to 5 g of 2'-deoxyadenine dissolved in
pyridine. 2.4 ml of Et3N and 1. 27 ml of MsCl are then
added to the mixture. After incubation for 2 h at
ambient temperature, the mixture is filtered and washed
with ethyl acetate. The filtrate is concentrated and
25 dissolved in 75 ml of ethanol, to which is added 1M of
NaOH. After refluxing for 1.5 h, the mixture is cooled
to ambient temperature and 1M of HCl is added. The
ethanol is evaporated off in a rotary evaporator and
the residue is extracted with CH2Cl2 • After silica gel
30 column purification, the product dATP-D1 is obtained.
Step D2: 2. 4 mmol of tert-butyldimethylsilyl chloride
are added to a stirred solution of 1.845 mmol of dATPwo
2016/034807 PCT/FR2015/052310
30
01 and 326
reaction is
temperature
applying a
mg of imidazole in anhydrous DMF. The
incubated with stirring at ambient
for 20 h. The solvents are removed by
vacuum and the residue is purified by
5 chromatography to give the product dATP-D2.
Step D3: The compound dATP-D2 is resuspended in
methanol and treated with aqueous concentrated
hydrochloric acid. The solution is cooled to -20°C
10 overnight, resulting in the product dATP-D3.
Step D4: 425 mg of 5'-0H nucleoside analog are treated
similarly to step A5 of example 1. The final mixture is
filtered and purified by reverse-phase HPLC to give a
15 triphosphate compound, in the case in point dATP-D4.
Step D5: 3.1 pmol of the compound dATP-D4 in 200 pl of
0 .1M NaHC03 , pH 8. 0, are mixed with 3. 4 )lmol of 2, 2-
tert-butyl-1-(2-nitro-4-biotin)phenyl)hexyl (2,5-dioxo-
20 pyrrolidin-1-yl) carbonate ln 200 )ll of
dimethylformamide. The reaction is carried
ambient temperature overnight to give dATP-D5
et al., PNAS, 1995, Vol 92, 7590-7594).
out at
(Olejnik
25 Step D6: 3.75 mmol of the compound dATP-D5 are
dissolved in 20 ml of THF and treated with 1M of TBAF
(tetra-n-butylammonium fluoride) in THF. The reaction
is complete after approximately 2 h with stirring. The
mixture is extracted with CH2Cl2 and purified by
30 chromatography to give dATP-NitroB-Biot.
Example 5 - Synthesis of the compound dCTP-NitroB-Biot
(fig 9)
5
- wo 2016/034807 PCT/FR2015/052310
31
Step E1: 2.2 ml of Et3N and 175 mg of DMAP and then
5.25 g of DMTCl are added, at ambient temperature,
overnight, to 5 g of 2'-deoxycytidine dissolved in
pyridine. 2.4 ml of Et3N and 1. 27 ml of MsCl are then
added to the mixture. After incubation for 2 h at
ambient temperature, the mixture is filtered and washed
with ethyl acetate. The filtrate is concentrated and
dissolved in 75 ml of ethanol, to which is added 1M of
10 NaOH. After refluxing for 1.5 h, the mixture is cooled
to ambient temperature and 1M of HCl is added. The
ethanol is evaporated off in a rotary evaporator and
the residue is extracted with CH2Cl2. After silica gel
column purification, the product dCTP-E1 is obtained.
15
Step E2: 2. 4 mmol of tert-butyldimethylsilyl chloride
are added to a stirred solution of 1.845 mmol of dCTPE1
and 326 mg of imidazole in anhydrous DMF. The
reaction is incubated with stirring at ambient
20 temperature for 20 h. The solvents are removed by
applying a vacuum and the residue is purified by
chromatography to give the product dCTP-E2.
Step E3: The compound dCTP-E2 is dissolved in absolute
25 ethanol and cooled to 0 °C. An equimolar solution of
2,2-tert-butyl-1-(2-nitro-4-biotin)phenyl)propylphenyl
carbonate in absolute ethanol is added dropwise. The
mixture is stirred at ambient temperature overnight.
The solution is filtered, washed with water and
30 extracted with CH2Cl2 to give dCTP-E3.
Step E4:
methanol
The compound dCTP-E3 is resuspended in
and treated with aqueous concentrated
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32
hydrochloric acid. The solution is cooled to -20°C
overnight, resulting in the product dCTP-E4.
Step E5: 425 mg of 5'-0H nucleoside analog are treated
5 similarly to step AS of example 1. The final mixture is
filtered and purified by reverse-phase HPLC to give a
triphosphate compound, in the case in point dCTP-E5.
Step E6: 3.75 mmol of the compound dCTP-E5 are
10 dissolved in 20 ml of THF and treated with 1M of TBAF
(tetra-n-butylammonium fluoride) in THF. The reaction
is complete after approximately 2 h with stirring. The
mixture is extracted with CH2Cl2 and purified by
chromatography to give dCTP-NitroB-Biot.
15
Example 6
(figure 16)
Synthesis of the compounds FA-Cat-dNTP
Step F1: 100 pl of 1M of modifier catechol ester in DMF
20 are mixed with 110 pl of 1M dicyclohexylcarbodiimide
(DCC) and 5 pl of 100% 4-(dimethylamino)pyridine in
DMF. The mixture is incubated at 0°C for 5 min. 500 pl
of 50 mM deoxyribonucleotide 5'-triphosphate in DMF are
then added to the mixture. The product is formed in 3 h
25 at ambient temperature. It is then precipitated with
acetone and dissolved in water so as to be finally
purified by chromatography to give the product FA-CatdNTP.
3 0 Deprotection
After the addition of the nucleotide to a nucleic acid
chain, the following examples 7 to 11 illustrate
wo 2016/034807 PCT/FR2015/052310
33
embodiments of the deprotection of said molecule, that
is to say the removal of the modifier group(s).
Example 7 - Deprotection of the polymerized nucleotides
5 NHrdT-Ni troB-Biot (fig 10)
The cleavage of the various modifier groups is carried
out simultaneously during the following process. 20 mM
of compound NH2-dT-NitroB-Biot in aqueous solution are
10 treated with a solution comprising 350 to 700 mM of
NaN02 and 1M NaOAc, pH 5.5. After incubation for 1 to
2 min at ambient temperature exposed to UV light having
a wavelength of 365 nm, the reaction is stopped by
adding 1M phosphate buffer, pH 7. 0, and stopping the
15 illumination. The deprotection reaction product is dT.
Example 8 - Deprotection of the polymerized nucleotides
NH2-dG-NitroN-Biot (fig 11)
20 The cleavage of the various modifier groups is carried
out simultaneously during the following process: 20 mM
of compound NH2-dG-NitroN-Biot in aqueous solution are
treated with a solution comprising 350 to 700 mM of
NaN02 and 1M NaOAc, pH 5.5. After 1 to 2 min of
25 incubation at ambient temperature exposed to UV light
having a wavelength of 365 nm, the reaction is stopped
by adding 1M phosphate buffer, pH 7.0, and stopping the
illumination. The deprotection reaction product is dG.
30 Example 9 - Deprotection of the polymerized nucleotides
of FA-Biot-dN type (fig 12)
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34
The cleavage of the modifier groups borne by the 3'-0H
end is carried out by hydrolysis of the ester function
with an aqueous ammonia solution, 1 to 100 mM, at
ambient temperature for 1 h. The product obtained is of
5 dN type.
Example 10 Deprotection of the polymerized
nucleotides dA-NitroB-Biot by photocleavage (fig 13)
10 The cleavage of the modifier groups borne on the
nitrogenous base is carried out by photocleavage. The
compound dA-NitroB--Biot is exposed to a. UV source
having a wavelength of 300 to 370 nm at ambient
temperature. The UV source is stopped after 30 to
15 300 seconds to give the product dA.
20
Example 11
nucleotides
(fig 14)
Deprotection of the polymerized
dC-NitroB-Biol by chemical cleavage
The cleavage of the modifier groups borne on the
nitrogenous base is carried out by chemical cleavage.
0. 01 mmol of compound dC-NitroB-Biot is dissolved in
0. 1 ml of ethanol. A solution of 0. 02 mmol of sodium
25 tetrachloropalladate II dissolved in ethanol is added.
Dihydrogen gas is bubbled into the mixture with
stirring for 20 min. The product obtained is dC.
A similar procedure is described by Kobayashi et al.
30 (Science, 2004, 304, 1305-1308) using immobilized
palladium, and a stream of H2 of 1 ml/min in THF can be
used as a variant.
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35
Example 12 Use of nucleotides according to the
invention for a nucleic acid synthesis
The modified nucleotides which are the subject of the
5 present invention can be advantageously used to carry
out the enzymatic synthesis of nucleic acids without
the presence of template strands according to the
process described in patent application FR 14-53455.
The enzyme chosen for carrying out the step of adding
10 the modified nucleotides is the commercially available
terminal deoxynucleotidyl transferase or TdT.
15
The primer used to initiate the synthesis is given
below:
15'-AGCCAAGCGGTCGCGATGAT-3'
The modified nucleotides used are NHrdTTP-Ni troB-Biot
prepared according to example 1. They make it possible
to add a T to the sequence No. 1 presented. It is
expected that only one nucleotide will be added to each
20 DNA fragment during each elongation step, as described
below.
25
A glass plate bearing "capture" fragments having the
following sequence:
5'-GTCCGCTTGGCT -3'
attached to this glass plate by their 3' end, is used
to capture the primers of sequence 1. This glass plate
constitutes the base of a parallelepipedal reaction
chamber 1 having a volume of 50 Jll. The t:apture is
30 carried out using a buffer solution comprising: 20 mM
Tris-HCl (pH= 7.5), 500 mM LiCl and 1 mM EDTA to which
wo 2016/034807 PCT/FR2015/052310
36
are added 2 pmol of primer. The capture step is carried
out over the course of 30 min at ambient temperature.
Once the primers have been captured, the plate is
washed by adding and removing three times 50 pl of the
5 following buffer solution: 20 mM Tris-HCl (pH = 7. 5),
200 mM LiCl and 1 mM EDTA.
The synthesis begins with the addition of the following
reagents to the reaction chamber: 50 U of TdT, 1 M of
10 potassium cacodylate, 125 mM of Tris-HCl, 0. 05% (v /v)
of Triton X-100, 5 mM of CoCl2 , at pH 7.2. Next are
added 100 pM of NHrdTTP-NitroB-Biot nucleotides which
are free and in solution with their counterions. The
enzyme at 2 pM is finally added in order to start the
15 addition reaction. The total reaction volume is 50 pl.
The mixture is incubated for 5 min at 37°C.
Once the · synthesis reaction has been completed, the
plate is washed 3 times with the following buffer
20 solution: 20 mM Tris-HCl (pH 7. 5), 200 mM LiCl and
1 mM EDTA. This has the effect of ensuring that the NH2 -
dTTP-Ni troB-Biot nucleotides introduced in excess are
removed, leaving the reaction chamber and the solid
support free of any unreacted nucleotide. At the end of
25 the washing, 50 pl of a 20 mM Tris-HCl buffer (pH =
7.5) are added to the reaction chamber and the
temperature is increased to 90 °C. This has the effect
of ensuring the detachment of the fragments having
incorporated the modified nucleotide NH2-dTTP-NitroB-
30 Biot. These fragments are collected and transferred
into a new Eppendorf tube.
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37
The DNA fragments having incorporated the protected
nucleotide NH2-dTTP-NitroB-Biot are then purified
according to the following procedure. Commercial
magnetic beads coated with streptavidin
5 (ThermoScientific) prepared according to the
manufacturer's protocol are added to the 50 pl of the
previous reaction mixture. After incubation for 1 h at
ambient temperature, the magnetic beads are collected
by means of a suitable magnet. The supernatant is then
10 removed. The beads are then washed 3 times with the
washing buffer: tris buffer, pH 7.2, with 0.1% of
Tween--20.
The magnetic beads to which the DNA fragments having
15 incorporated the modified nucleotides NH2-dTTP-NitroBBiot
are attached are resuspended in a solution
comprising 350 to 700 mM of NaN02 and 1M NaOAc at pH
5.5. The mixture is incubated for 1 to 2 min at ambient
temperature under exposure to UV (365 nm). The reaction
20 is stopped by adding 1M phosphate buffer at pH 7.0 and
stopping the illumination. This operation enables the
"detachment" of the DNA fragments from their supports
(beads).
25 The magnetic beads are collected by means of a suitable
magnet. The supernatant is recovered and analyzed by
electrophoresis gel and MALDI -TOF MS spectrometer in
order to verify the correct incorporation of the T base
at the 3' end of the sequence No. 1 in more than 99% of
30 cases.
A new elongation step may then be carried out if
necessary, according to the same protocol.
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38
Example 13 Other example of use of nucleotides
according to the invention for a nucleic acid synthesis
5 The modified nucleotides which are the subject of the
present invention can be advantageously used to carry
out the enzymatic synthesis of nucleic acids without
the presence of template strands according to the
process described in patent application FR 14-53455.
10 The enzyme chosen for carrying out the step of adding
the modified nucleotides is the commercially available
terminal deoxynucleotidyl transferase or TdT.
The primer used to initiate the synthesis is given
15 below:
5'-AGCCAAGCGGTCGCGATGAT~3' Seq No1
The modified nucleotides used are NHrdGTP-Ni troB-Biot
prepared according to Example 2. They make it possible
20 to add a G to the sequence No. 1 presented. It is
expected that only one nucleotide will be added to each
DNA fragment during each elongation step, as described
below.
25 A glass plate exhibiting capture fragments having the
following sequence:
5'~GTCCGCTTGGCT-3' Seq N°2
and attached to this glass plate by their 3' end, is
used to capture the primers of sequence 1. This glass
30 plate constitutes the base of a parallelepipedal
reaction chamber having a volume of 50 pl. The capture
is carried out using a buffer solution comprising: 20
wo 2016/034807 PCT/FR2015/052310
39
mM Tris-HCl (pH 7.5), 500 mM LiCl and 1 mM EDTA to
which are added 2 pmol of primer.
carried out over the course of
The capture step is
30 min at ambient
temperature. Once the primers have been captured, the
5 plate is washed by adding and removing 3 times 50 pl of
the following buffer solution: 20 mM Tris-HCl (pH
7.5), 200 mM LiCl and 1 mM EDTA.
The synthesis begins with the addition of the following
10 reagents to the reaction chamber: 50 U of TdT, 1M of
potassium cacodylate, 125 mM of Tris-HCl, 0. 05% (v/v)
of Triton X-100; 5 mM of CoC12 , pH 7.2. Next are added
100 pM of nucleotides NH2-dGTP-NitroB-Biot which are
free and in solution with their counterions. The enzyme
15 at 2 pM is finally added in order to start the addition
reaction. The total reaction volume is 50 pl. The
mixture is incubated for 5 min at 37°C.
Once the synthesis reaction is complete, the plate is
20 washed 3 times with the following buffer solution:
20 mM Tris-HCl (pH = 7.5), 200 mM LiCl and 1 mM EDTA.
This has the effect of ensuring that the NH2-dGTPNitroB-
Biot nucleotides introduced in excess are
removed, leaving the reaction chamber and the solid
25 support free of any unreacted nucleotide. At the end of
the washing, 50 pl of a 20 mM Tris-HCl buffer (pH =
7.5) are added to the reaction chamber and the
temperature is increased to 90 °C. This has the effect
of ensuring the detachment of the fragments having
30 incorporated the modified nucleotide NH2-dGTP-NitroBBiot.
These fragments are collected and transferred
into a new Eppendorf tube.
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40
The DNA fragments having incorporated the protected
nucleotide NH2-dGTP-NitroB-Biot are then purified
according to the following procedure. Commercial
magnetic beads coated with streptavidin
5 (ThermoScientific) prepared according to the
manufacturer's protocol are added to the 50 pl of the
previous reaction mixture. After incubation for 1 h at
ambient temperature, the magnetic beads are collected
by means of a suitable magnet. The supernatant is then
10 removed. The beads are then washed 3 times with the
washing buffer solution: tris buffer, pH 7.2, with 0.1%
of Tween 20.
The magnetic beads to which the DNA fragments having
15 incorporated the modified nucleotides NHrdGTP-Ni troBBiot
are attached are resuspended in a solution
comprising 350 to 700 mM of NaN02 and 1M NaOAc at pH
5.5. The mixture is incubated for 1 to 2 min at ambient
temperature with exposure to UV (365 nm) . The reaction
20 is stopped by adding 1M phosphate buffer, pH 7.0, and
stopping the illumination. This operation allows the
"detachment" of the DNA fragments from their supports
(beads).
25 The magnetic beads are collected by means of a suitable
magnet. The supernatant is recovered and analyzed by
electrophoresis gel and MALO I -TOF MS spectrometer in
order to verify the correct incorporation of the T base
at the 3' end of the sequence No. 1 in more than 99% of
30 cases.
If necessary, a new elongation step can then be carried
out according to the same protocol.
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41
Industrial application
The modified nucleotides which are subjects of the
5 present invention improve the performance levels of
10
nucleic acid synthesis processes by allowing in
particular the synthesis of very long nucleic acids of
very high quality. These nucleotides can be used for
the production, on a more or less large scale, of
synthetic nucleic acid sequences or genes. These
modified nucleotides are particularly intended for the
synthesis of nucleic acids such as DNA or RNA for
research, development or industrialization purposes in
the biotechnology field or more generally in the broad
15 field of biology.

CLAIMS
1. A modified nucleotide, intended for the enzymatic
synthesis of nucleic acids, comprising a "natural"
5 nitrogenous base or a natural nitrogenous base analog,
a ribose or deoxyribose carbohydrate, and at least one
phosphate group,
characterized in that it comprises at least one R or R'
group, called modifier group, borne:
10 - by said natural nitrogenous base or analog,
- and/or by the oxygen in position 3' of the ribose or
deoxyribose molecule, making it possible to block the
polymerization of said nucleotide and/or to allow the
interaction of said nucleotide with another molecule,
15 different than another nucleotide, such as a protein,
during nucleic acid synthesis, R comprising at least
one functional end group, said nucleotide being in the
form of one of formulae (I), (III) and (IV) below:
(}) R
/--(oy•L
(PI')PO !-I ( OH)
z
R' I (M}
l
0 (Formula I)
20
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43
(Formula III)
0
+ (Formula IV)
5 in which:
(PP)PO represents a mono-, di- or triphosphate group,
(OH) describes the possibility of a ribose or
deoxyribose molecule,
T is a hydrogen, or a cleavable radical chosen from
10 -NH2, -N3, - (C=O) H, -CnH2n+l with n between 1 and 30,
preferably between 1 and 12, -trimethylsilyl,
-phosphate, -S03 , -(C=O)OCnH2n+l with n between 1 and 30,
preferably between 1 and 12, - ( C=O) SCnH2n+l with n
between 1 and 30, preferably between 1 and 12,
15 -nitrobenzene, -benzyl, -halobenzyl, -amide,
-carbonate, -benzoyl, -peroxyl, -nitrile, -thiol,
wo 2016/034807
-imide, -carbamate,
-halophenyl, -picolyl,
44
-cyanate,
PCT/FR2015/052310
-alkyne, -phenyl,
M, which is optionally present, is a group covalently
bonded to Q and to Z, M being chosen from alkyl,
5 alkenyl, alkyne, aryl, alkylaryl, heteroaryl, acyl,
alkyloxy, alkylamino, alkoxyamino, amido, alkylimido,
alkenylimido, arylimido, fluoroalkyl, alkylphosphate,
alkyl thio, thioacyl, alkylsulfonyl, aryl sulfonyl,
alkylsulfinyl, alkylammonium, alkylsulfonium,
10 alkylsilyl, alkylcarbonyl, alkylcarbanyl,
alkylcarbamoyl or alkylhydroxylamino,
Z is a cleavable group, chosen from -0-, -S-, =SH-,
=S=, =S-, -SiH2-, =SiH-, =Si=, =Si-, -Se-, =SeH-, =Se-,
=Se=, -SeH2-, -PH-, =P-, =PH=, =P=, =PH-, -PH3-, -AsH-,
15 =As-, =AsH=, =As=, =AsH-, -ASH3-, amine, ester, silyl,
alkyl, benzyl, nitrobenzyl, amide, carbonate, benzoyl,
peroxyl, nitrile, thiol, imide, carbamate, cyanate,
hydroxylamine, sulfoxide, sulfonate, thiosulfinate,
thioester, acyl halide, hypoiodyl, alkyne, halophenyl,
20 halobenzyl, picoyl, diol or disulfide, or chosen from
-CH2 or -NH- when M is a -nitrobenzyl-, a -nitrotolyl-,
a -nitroxylyl-, a -nitronaphthyl- or a -nitrophenyl-,
Q is an end functional, or effector, group of the R or
R' group, Q being chosen from biotin, a protein, a
25 polynucleotide of defined sequence, a carbohydrate, an
antigen, a hormone, a neurotransmitter, a glycoside
such as digoxin, a sulfur-containing radical, in
particular bearing a thiol function, such as
glutathione, or a bidentate ligand such as catechol,
30 R and R' possibly being present independently or
simultaneously, and when R and R' are present
simultaneously:
the z groups may be identical or different,
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45
the M groups may be identical or different,
the Q groups may be identical or different,
"base" represents a "natural" nitrogenous base chosen
from adenine, thymine, cytosine, guanine or uracil or a
5 natural nitrogenous base analog, with the exception of
thymine when R' is present and Q comprises biotin.
2. The nucleotide as claimed in claim 1, characterized
in that the modifier group R is borne by the
10 nitrogenous base and forms one of the structures (V)
below:
HN-Z-M-Q 0
3
6 ~N~-~Z-M-Q
/N Sugar
1
· 0
Adenine-based structure (Va) Thymine-based structure (Vt)
7
HN-Z-M-0 .KN,
>~ Sugar o
·rN
9 N
/
Sugar
15 Cytosine-based structure (V0 ) Guanine-based structure (Vg)
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46
Uracil-based structure (Vu)
in which structures:
"sugar" represents the bond between said nitrogenous
5 base and the ribose or deoxyribose molecule of the
nucleotide molecule,
Z1 and Z2 are identical or different, cleavable Z
groups.
10 3. The nucleotide as claimed in claim 2, characterized
in that the modifier group R borne by the nitrogenous
base forms one of the structures (VI) below: H----H->-Q
s /~--<3---z
Sugar o
Adenine-based structure (VIa) Thymine-based structure (VId
HN Z J O----H-X1 ;Q), ,\_Q
Sugar
4 ~~ I
Sugar o~~~*H-x2 HN Z2
15
Cytosine-based structure (VI0 ) Guanine-based structure (VIg)
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47
0-- --H--X1 /! [! \ s.t;---!/ ~>.1-o /I 4\3 / 6\ .~ z
./~ 2< Sugar' ~0 Uracil-based structure (VIu)
in which:
"Sugar" represents the bond between the nitrogenous
base and the ribose or deoxyribose molecule of the
5 nucleotide molecule,
which may be identical or different,
represent nitrogen, oxygen or sulfur atoms borne by M
and capable of forming, with said nitrogenous bases of
the modified nucleotide, intermolecular hydrogen bonds
10 (similar to those observed during conventional pairings
between complementary nucleotides).
4. The nucleotide as claimed in one of the preceding
claims, which can be used as a substrate for
15 polymerases that are normally dependent on the presence
of a template nucleic acid strand complementary to the
strand undergoing synthesis, even in the absence of a
complementary strand.
20 5. The nucleotide as claimed in any one of the
preceding claims, characterized in that the functional
end radical Q of the R or R' group is capable of
interacting with molecules other than a nucleic acid
according to one or another of the following
25 interaction pairs: antigen/antibody, hormone/receptor,
biotin/(strept)avidin,
polymerase/promoter,
neurotransmitter/receptor,
digoxin/antidigoxin,
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48
carbohydrate/lectin, sulfur-containing radical/metal
such as gold, glutathione/glutathione S-transferase,
bidentate ligand/metal oxide.
5 6. The nucleotide as claimed in claim 3, for which X1
and X2 are -NH, T is -NH2, Z is
methylnitrobenzyl-, and Q is -biotin.
M is
7. The nucleotide as claimed in claim 3, for which X1
10 and X2 are -NH, T is -NH2, Z, Z1 and Z2 are each -0-, M
is -nitronaphthyl- and Q is -biotin.
8. The nucleotide of formula (I) as claimed in claim 1,
bearing only the R' group in which: Z is -(C=O)-, M is
15 -C8H16- and Q is -NH-biotinyl.
9. The nucleotide as claimed in claim 2, characterized
by the particular structure (V) in which Z, Z1 and Z2
are each -(COO)-, M is -tert-butylnitrobenzyl- and Q is
20 -NH-biotinyl.
25
10. The nucleotide as claimed in any one of the
preceding claims, characterized in that T and Z, or Z1 ,
Z2 are cleavable, during the nucleic acid synthesis, by
irradiation of said nucleotide by means of
electromagnetic radiation having a wavelength of
between 10-3 and 10-11 meter, in particular by
ultraviolet radiation.
30 11. The use of a nucleotide as claimed in any one of
the preceding claims, in a process for the production
of genes, of synthetic nucleic acid sequences, of DNA,
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49
of RNA or of nucleic acid polymers, in particular·
according to an enzymatic synthesis process.
12. The use of a nucleotide as claimed in any one of
5 the preceding claims, for incorporating said nucleotide
into a polynucleotide chain previously immobilized on a
solid support.
13. · The use of a nucleotide 9-s claimed in claim 12,
10 characterized in that the polynucleotide chain is
attached via its 5' erld and the incorporation of said
nucleotide is carried out via the 3' end of the
polynucleotide chain.
15 14. A kit for nucleic acid synthesis, comprising at
least one modified nucleotide as claimed in any one of
the preceding claims.
15. The kit as claimed in claim 14 in combination with
2 0 claim 12} comprising various modified nucleotides, an
elongation enzyme and a solid support capable ·of
attaching at least ohe of said nucleotides.