U.S. patent application number 14/748930 was filed with the patent office on 2016-01-28 for method for deblocking of labeled oligonucleotides.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Gulilat GEBEYEHU, Richard Pires.
Application Number | 20160024138 14/748930 |
Document ID | / |
Family ID | 22469987 |
Filed Date | 2016-01-28 |
United States Patent
Application |
20160024138 |
Kind Code |
A1 |
GEBEYEHU; Gulilat ; et
al. |
January 28, 2016 |
METHOD FOR DEBLOCKING OF LABELED OLIGONUCLEOTIDES
Abstract
The invention relates to a process for deblocking substantially
a blocked, detectably labeled oligonucleotide by contacting the
blocked detectably labeled oligonucleotide with an effective amount
of a nucleophilic amino compound under conditions that result in
substantial deblocking of the oligonucleotide, thereby giving the
substantially deblocked oligonucleotide.
Inventors: |
GEBEYEHU; Gulilat; (Potomac,
MD) ; Pires; Richard; (Mount Airy, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
22469987 |
Appl. No.: |
14/748930 |
Filed: |
June 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13964036 |
Aug 9, 2013 |
9085797 |
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14748930 |
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13152907 |
Jun 3, 2011 |
8524882 |
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13964036 |
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10365644 |
Feb 13, 2003 |
8093372 |
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13152907 |
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09576555 |
May 24, 2000 |
6593464 |
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10365644 |
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60135848 |
May 24, 1999 |
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Current U.S.
Class: |
536/25.31 |
Current CPC
Class: |
C07B 2200/11 20130101;
C12Q 1/68 20130101; C07H 21/00 20130101 |
International
Class: |
C07H 21/00 20060101
C07H021/00 |
Claims
1. A process for deblocking substantially a blocked, detectably
labeled oligonucleotide comprising contacting the blocked
detectably labeled oligonucleotide with an effective amount of a
nucleophilic amino compound under conditions that result in the
deblocking of the oligonucleotide, thereby giving the substantially
deblocked oligonucleotide.
2. The process of claim 1, wherein said detectable label is a
fluorescent label.
3. The process of claim 2, wherein said detectable label is
hexachlorofluorescein.
4. The process of claim 2, wherein said detectable label is
DABCYL.
5. The process of claim 1, wherein said nucleophilic amino compound
is ammonia.
6. The process of claim 5, wherein said ammonia is present at a psi
of about 20 to 200.
7. The process of claim 5, wherein said ammonia is present at a psi
of about 80.
8. The process of claim 1, wherein said nucleophilic amino compound
is ammonia vapors.
9. The process of claim 1, wherein said nucleophilic amino compound
is a C.sub.16 alkylamine.
10. The process of claim 1, further comprising dissolving the
substantially deblocked oligonucleotide in a buffer.
11. The process of claim 1, wherein said conditions comprise
carrying the process at about room temperature to about 150.degree.
C.
12. The process of claim 1, wherein said conditions comprise
carrying the process at about 95.degree. C.
13-14. (canceled)
15. The process of claim 1, wherein said substantially blocked,
detectably labeled oligonucleotide is immobilized on a solid
phase.
16. The process of claim 15, wherein said substantially deblocked
oligonucleotide is released from said solid phase under said
conditions.
17. The process of claim 16, wherein said substantially deblocked
oligonucleotide is recovered by washing said solid phase with water
or a buffer.
18. A process for deblocking substantially a blocked, detectably
labeled oligonucleotide comprising contacting the blocked
detectably labeled oligonucleotide with an effective amount of
ammonia saturated with water vapor at about 80 psi, 95.degree. C.
for about 45 min, thereby giving the substantially deblocked
oligonucleotide.
19-21. (canceled)
22. A composition comprising a blocked, detectably labeled
oligonucleotide and an effective amount of a nucleophilic amino
compound sufficient to deblock substantially said
oligonucleotide.
23. The composition of claim 22, wherein said detectable label is a
fluorescent label.
24. The composition of claim 22, wherein said nucleophilic amino
compound is ammonia.
25. The composition of claim 22, wherein said nucleophilic amino 15
compound is ammonia vapors.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/135,848, filed May 24, 1999, the
contents of which are entirely incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to processes for the
substantial deprotection or deblocking of labeled oligonucleotides
by use of an amino reagent such as ammonia.
[0004] 2. Related Art
[0005] A variety of solid phase oligonucleotide synthesis
techniques are known to those skilled in the art. Such techniques
include phosphoramidite, phosphotriester, phosphodiester, phosphite
and H-phosphonate methods and the like, each of which is generally
known in the field of molecular biology. For example, the
b-cyanoethyl phosphoramidite method is described in U.S. Pat. No.
4,458,066 issued to Caruthers, et al., entitled "Process for
Preparing Polynucleotides," which is incorporated herein by
reference.
[0006] The phosphoramidite based synthesis of oligonucleotides
requires the protection of the exocyclic amino groups. The most
commonly used protecting groups for this purpose are benzoyl for
the 6-amino of adenine and 4-amino of cytosine and isobutyroyl for
2-amino of guanine. Oligonucleotides are synthesized on solid
support using nucleoside phosphoramidites where the amino groups
are protected as shown below.
##STR00001##
[0007] After the synthesis is completed the oligonucleotide is
cleaved from the support and these protecting groups are removed by
hydrolysis at high temperatures using concentrated ammonium
hydroxide. After hydrolysis, the ammonium hydroxide has to be
evaporated in order to obtain the desired oligonucleotide.
[0008] The use of hot concentrated ammonium hydroxide for the
removal of these protecting groups has restricted the modified
bases that can be used to those that can withstand these harsh
conditions. For modified oligos such as dye containing oligos,
heating in ammonium hydroxide can not be used since the dyes are
not stable under these conditions. In general, dye labeled oligos
arc deprotected by treatment with ammonium hydroxide at room
temperature for over 24 hours or require special reagents for this
purpose. See U.S. Pat. No. 4,965,349. Alternatively, oligos may
prepared using phosphoramidite having easily removable protecting
groups which do not require the use of hot concentrated ammonium
hydroxide. See Boal, J. H. et al., Nucl. Acids Res. 24:3115-3117
(1996).
[0009] The standard method where deprotection with concentrated
ammonium hydroxide is done at reduced temperature results in
incomplete deprotection and over all low quality of dye labeled
oligonucleotides. Often this requires tedious purification which
results in low yield.
[0010] U.S. Pat. No. 4,965,349 describes a method of hydrolyzing
base-labile linking groups between a solid phase support and
oligonucleotides with a reagent comprising a lower alcohol, water
and a non-nucleophilic hindered alkylamine.
[0011] According to this patent, this cleavage reagent preserves
the fluorescent characteristics of rhodamine dyes during cleavage
from the solid support.
[0012] U.S. Pat. No. 5,514,789 describes a method for the cleavage
and deprotection of newly synthesized oligonucleotides from solid
supports with a gaseous cleavage/deprotection reagent such as
gaseous ammonia, ammonium hydroxide vapors, and methylamine.
[0013] U.S. Pat. No. 5,518,651 describes a method for the cleavage
and deprotection of insolubilized and protected oligonucleotides
using an alkyl amine, e.g. t-butylamine and methylamine. According
to this patent, the deprotection and cleavage of the
oligonucleotides occurs at room temperature and in less than about
90 min.
[0014] U.S. Pat. No. 5,738,829 describes a method for the cleavage
and deprotection of oligonucleotides from solid supports which
involves incubation of the immobilized oligonucleotides with
gaseous ammonia or ammonium hydroxide vapors. According to this
patent, the method lends itself to the use of supports such as
microtiter plates that can be used to perform up to 96 individual
synthetic processes.
[0015] Glenn Research of Sterling Virginia offers phenoxyacetyl
protected dA, 4-isopropylphenoxylacetyl protected dG and acetyl
protected dC which can be used to prepare oligonucleotides.
According to Glenn Research's web site, these monomers can be used
with sensitive labeling reagents such as TAMRA, Cy5.RTM. and HEX
since cleavage and deprotection can be carried out in 2 hours at
room temperature with ammonium hydroxide or 0.005 M potassium
carbonate in anhydrous methanol. In addition, according to this web
site, it is possible to deprotect oligonucleotides containing
acetyl protected dC monomers by treatment with ammonium
hydroxide/methylamine for 10 min at 65.degree. C. or less.
[0016] We have now found that the use of nucleophilic amino
compounds under pressure and high temperature is an effective way
to deprotect dye labeled oligos. The dye labeled oligos deprotected
in this manner are fully deprotected and are of high quality. In
addition, the process is simple and saves time, reducing the
deblocking (processing) time from approximately 28 hours to 1
hour.
BRIEF SUMMARY OF THE INVENTION
[0017] The invention relates to a process for deblocking a
detectably labeled oligonucleotide comprising contacting the
blocked detectably labeled oligonucleotide with an effective amount
of a nucleophilic amino compound under conditions that result in
the deblocking of the oligonucleotide, thereby giving the deblocked
oligonucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A and 1B depict ion-pair HPLC chromatograms showing
the analysis of the dye labeled oligo (5'-FAM-GGT CCG ACC AGA TGG
CGA AAG GCA AAC GGA; SEQ ID NO:1) after deprotection with
concentrated ammonium hydroxide, RT, 24 hrs (FIG. 1A) or with
gaseous ammonia (80 psi), 95.degree. C., 45 min (FIG. 1B). Buffer
A=5 mM TBAP in 20 mM NH.sub.4HPO.sub.4. Buffer B=CH.sub.3CN,
gradient of 40% B to 60% B over 10 min, flow rate of 2 ml/min.
[0019] FIGS. 2A-2D depict ion-pair HPLC chromatograms showing the
analysis of molecular beacon (5'-HEX-GCG ACG CCT GTC CTC CAA TTT
GTC CTG GTC GTC GC DABCYL) after deprotection with concentrated
ammonium hydroxide, 90.degree. C., 75 min (FIG. 2A), concentrated
ammonium hydroxide, RT, 24 hrs (FIG. 2B), concentrated ammonium
hydroxide, RT, 40 hrs (FIG. 2C), or with gaseous ammonia (80 psi),
95.degree. C., 1 hr (FIG. 2D). Buffer A=5 mM TBAP in 20 mM
NH.sub.4HPO.sub.4. Buffer B=CH.sub.3CN, gradient of 40% B to 60% B
over 10 min, flow rate of 2 ml/min.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The invention relates to a process for deblocking
substantially detectably labeled oligonucleotides comprising
contacting the blocked detectably labeled oligonucleotide with an
effective amount of a deblocking reagent such as a nucleophilic
amino compound. Preferably, the deblocking reagent is gaseous at
ambient temperature.
[0021] The present invention provides a number of advantages over
conventional methods of deblocking oligonucleotides using aqueous
ammonia, including improved quality (purity) of the deblocked
oligonucleotide, higher yield, and shorter reaction times.
Moreover, the amino compound may be removed substantially by
degassing (in the case of ammonia, methylamine and ethylamine),
thus providing ease of recovery of the deblocked oligonucleotide.
In the case of higher molecular weight amino compounds that are
liquids at room temperature, the amino compound may be removed by
washing the deblocked oligonucleotide with an organic solvent in
which the oligonucleotide is poorly soluble, e.g. acetonitrile,
diethyl ether and the like, to remove the amino compound. The
deblocked oligonucleotide may then be resuspended directly in
water, a buffer or other solution and used directly as a molecular
biology reagent, e.g. as a diagnostic reagent in sequencing, PCR or
as a probe. The buffer may be chosen to neutralize any residual
nucleophilic amino compound (e.g. ammonia) that may be present.
Particularly preferred buffers are the acetate, sulfate,
hydrochloride, phosphate or free acid forms of
Tris-(hydroxymethyl)amino-methane (TRIS.RTM.), although alternative
buffers of the same approximate ionic strength and pKa as TRIS.RTM.
may be used with equivalent results. Other preferred buffers are
triethyl ammonium salts (e.g. the acetate salt). In addition to the
buffer salts, cofactor salts such as those of potassium (preferably
potassium chloride or potassium acetate) and magnesium (preferably
magnesium chloride or magnesium acetate) may be included. Addition
of one or more carbohydrates and/or sugars to the buffer solution
and/or deblocking reaction mixtures may also be advantageous, to
support enhanced stability of the product upon storage. Preferred
such carbohydrates or sugars include, but are not limited to,
sucrose, trehalose, and the like. Such carbohydrates and/or sugars
are commercially available from a number of sources, including
Sigma (St. Louis, Mo.).
[0022] The nucleophilic amino compound may be ammonia or ammonia
vapors (e.g. obtained by heating a sealable chamber having a
quantity of ammonium hydroxide in the bottom), or a C.sub.1-6
alkylamino compound. The alkyl group may be straight or branched
chain. Examples of such alkylamino compounds include methylamine,
ethylamine, propylamine, isopropylamine, butylamine,
sec-butylamine, pentylamine and hexylamine. When the nucleophilic
amino compound is a liquid at ambient temperature, it may be
removed under vacuum with or without heating. In a preferred
embodiment, the nucleophilic amino compound is at least saturated
with water vapor.
[0023] The label on the oligonucleotide may be any conventional
label used for detection of oligonucleotides, including, without
limitation, fluorescent dyes chosen from the group consisting of
xanthenes (e.g., fluoresceins, eosins, erythrosins), rhodamines
(e.g., Texas Red.RTM.), benzimidazoles, ethidiums, propidiums,
anthracyclines, mithramycins, acridines, actinomycins,
merocyanines, coumarins (e.g., 4-methyl-7-methoxycoumarin),
pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes (e.g.,
dansyl, 5-dimethylamino-1-naphthalenesulfonyl), salicylic acids,
benz-2-oxa-1-diazoles (also known as benzofurans) (e.g.,
4-amino-7-nitrobenz-2-oxa-1,3-diazole), indodicarbocyanines (e.g.
Cy3.RTM. and Cy5.RTM., available from Biological Detection Systems,
Inc.), fluorescamine, and psoralen. See U.S. Pat. Nos. 4,997,928,
5,262,536, and EP 63,879. Useful forms of many of these dyes are
commercially available. See also A. W. Wagner, Chapter 1,
Applications of Fluorescence in the Biomedical Sciences, Taylor et
al. (ed.), Alan R. Liss, New York (1986). Particular examples
include 6-(fluorescein-6-carboxamido)hexanoate (6-FAM), fluorescein
isothiocyanate (FITC), hexachlorofluorescein (HEX),
tetrachlorofluorescein (TET),
6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein (6-JOE), and
BODIPY. In a preferred embodiment, the oligonucleotides is labeled
with the molecular beacon technology according to Tyagi, S. and
Kramer, E R., Nature Biotechnology 14:303-308 (1996). One label
that degrades in the presence of ammonia is (6-tetramethylrhodamine
(TAMRA).
[0024] The blocked, labeled oligonucleotides may be prepared by
well known methods, e.g. the phosphoramidite, phosphotriester,
phosphodiester, phosphite and H-phosphonate methods, each of which
are generally known in the field of molecular biology. For example,
the b-cyanoethyl phosphoramidite method is described in U.S. Pat.
No. 4,458,066 issued to Caruthers, et al., entitled "Process for
Preparing Polynucleotides," which is incorporated herein by
reference. See also E. Eckstein (ed.), Oligonucleotides and
Analogs, A Practical Approach, IRL Press, Oxford (1991); GB
2,125,789; and U.S. Pat. Nos. 4,415,732, 4,739,044 and 4,757,141.
Such oligonucleotides may be DNA, RNA, mixture of DNA and RNA,
derivatives of DNA and RNA and mixtures thereof. In the case of
RNA, base stable 2'-protecting groups are preferred. The blocked,
labeled oligonucleotide may be free or immobilized on a solid phase
which is also cleaved by the gaseous deblocking reagent. The
deblocked oligonucleotide may then be recovered by washing the
solid phase with water or a buffer.
[0025] The blocking group is present on the exocyclic amino groups
of A, G and C. Thymine does not require protection. Such blocking
groups may be C.sub.1-6 alkanoyl (e.g. isobutyryl), aryloyl
(benzoyl), phenoxyacetyl, C.sub.1-6 alkoxyacetyl, and dimethyl
formamidine (on N.sup.6 of dA or N.sup.2 of dG). When the
deblocking reagent is an alkylamine, it is preferred that the base
C be blocked with an acetyl group. The blocking group for the
phosphorous may be a cyanoethyl group. All of these blocking groups
are cleaved at the same time by the deblocking reagent.
[0026] The deblocking reaction is preferably carried out in a
sealable chamber (although an open chamber may be used in
accordance with the invention) that can be heated. Such sealable
chambers include screw cap vials, Parr bottles, and the like. The
oligonucleotide synthesis and cleavage from the support may be
carried out with a commercially available DNA synthesizer, e.g. the
ABI 380B DNA synthesizer, or other equipment that is set up for
high throughput synthesis on a multi well channel, e.g. a 96 well
plate (see, e.g., U.S. Pat. Nos. 5,472,672 and 5,529,756, and U.S.
application Ser. No. 09/162,348, filed Sep. 28, 1998, which are
incorporated herein by reference in their entireties).
[0027] The deblocking reagent is present in an amount effective to
deblock the oligonucleotide. In general, the deblocking reagent is
present in a large excess compared to the oligonucleotide. In the
case of ammonia, the sealable chamber may be charged with about 20
to 200 psi of ammonia, most preferably, about 80 psi. Optimal
amounts of the liquid alkylamino compounds may be determined with
no more than routine experimentation.
[0028] The deblocking reaction is carried out at a temperature of
about room temperature to about 150.degree. C. Most preferably,
when the deblocking reagent is ammonia, the reaction is carried out
at about 95.degree. C.
[0029] The deblocking reaction is carried out for about 1 min to
about 2 hrs. More preferably, the reaction is carried out for about
1 min to about 1 hr. When the deblocking reagent is ammonia, it is
preferred that the reaction be carried out for about 45 min.
[0030] By "substantially deblocked" is intended to mean that the
blocked oligonucleotide is not detectable, e.g. by ion-pair HPLC,
capillary electrophoresis or mass spectrometry, after the
deblocking reaction according to the present invention.
[0031] The following examples are illustrative, but not limiting,
of the method and compositions of the present invention. Other
suitable modifications and adaptations of the variety of conditions
and parameters normally encountered in molecular biology and
chemistry, particularly oligonucleotide synthesis, which are
obvious to those skilled in the art in view of the present
disclosure are within the spirit and scope of the invention.
EXAMPLES
[0032] An example of deblocking of a fluorescein (FAM) labeled
oligo under standard conditions (room temperature and 24 hrs) and
also gas phase (45 min, 80 psi, 95.degree. C.) is attached (FIGS.
1A and 1B). As can be seen from the chromatogram, the peak for the
standard deblocking is broader indicating that there is an
incomplete deprotection. However, the chromatogram deblocking with
ammonia shows a much sharper peak which is indicative of complete
deprotection and no degradation.
[0033] In another example an oligonucleotide labeled with
hexachlorofluorescein (HEX) and DABCYL (molecular beacon) was
deblocked under various conditions (FIGS. 2A-2D). Standard
deblocking conditions (95.degree. C., concentrated ammonia, 75 min,
FIG. 2A) as well as room temperature treatment over 24 hours (FIG.
2B and FIG. 2C) show formation of a new peak which may be
attributed to degradation of the dye. As can be seen from the
chromatographs (FIG. 2D) deblocking under gas phase gives better
quality oligonucleotides.
[0034] Experimental Procedure:
[0035] Place the dye-oligo-CPG (obtained from phosphoramidite based
automated synthesis) in a high pressure reactor. Charge the reactor
with gaseous, ammonia saturated with water vapor (80 psi pressure).
Heat the sealed reactor to 95.degree. C. for 45 min. Release the
pressure and cool the CPG to room temperature. Elute the
oligonucleotide from the CPG with water (.about.300 .mu.l is
sufficient for 50-200 nmol scale synthesis) and proceed to analysis
and purification.
[0036] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
in the art to which the invention pertains. All publications,
patents and patent applications are herein incorporated by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference in their entirety.
Sequence CWU 1
1
2130DNAArtificialOligonucleotide 1ggtccgacca gatggcgaaa ggcaaacgga
30235DNAArtificialOligonucleotide 2gcgacgcctg tcctccaatt tgtcctggtc
gtcgc 35
* * * * *