Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotides

The present invention provides novel compounds and methods for purifying oligonucleotides. The compounds according to the invention are multimeric oligonucleotides comprising a multimerization domain for inducing multimeric oligonucleotide aggregation, a hybridization domain that is complementary to a target oligonucleotide whose isolation is desired, and a linker domain connecting the multimerization domain and the hybridization domain. Other compounds of the invention comprise dendrimers having oligonucleotides with hybridization domains linked thereto. The methods of the invention comprise contacting the compounds of the invention with a solution containing a target oligonucleotide whose purification is desired. The target oligonucleotide hybridizes to the hybridization domain of the inventive compounds, thereby forming an aggregate. Synthetic failure sequences (N-1, N-2, etc.) and other oligonucleotides not complementary to the hybridization domain do not hybridize with the hybridization domain of the compounds and remain free in solution. Conventional size exclusion chromatography or small pore filter membranes are then used to separate the aggregate (and hence target oligonucleotide) from the other oligonucleotides. The aggregate is denatured and the target oligonucleotide separated once again by size exclusion chromatography or with a small pore filter membrane.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the field of purification of synthetic 
oligonucleotides. 
2. Summary of the Related Art 
Recent advances in the chemical synthesis of nuclease-resistant 
oligonucleotides (Agrawal, Trends in Biotechnol 10, 152 (1992); Agrawal et 
al., Opin. Biotechnol. 6, 12 (1995)), large scale solid phase synthesis 
(Padmapriya et al., Antisense Res. Dev. 185, 4 (1994)), and purification 
and analytical techniques (Methods in Molecular Biology, Vol 20, Protocols 
for Oligonucleotides and Analogs (Agrawal, Ed., Humana Press, Totowa, 
N.J., 1993); Methods in Molecular Biology, Vol 26, Protocols for 
Oligonucleotide Conjugates (Agrawal, Ed., Humana Press, Totowa, N.J., 
1994)) have permitted rational design of sequence specific antisense 
oligonucleotides and their advancement to human clinical trails (Zhang et 
al., Clin. Pharmacol. Ther. 58, 44 (1995); Iversen et al., Antisense Res. 
Dev. 4, 43 (1994); Crooke et al., Clin. Pharmacol. Ther. 56, 641 (1994)). 
Reverse-phase HPLC, ion exchange chromatography, and gel electrophoresis 
are currently used for oligonucleotide purification. Methods in Molecular 
Biology, Vol. 26, supra. 
Conventional methods of purification rely on such oligonucleotide 
characteristics as charge and hydrophobicity/hydrophilicity. Consequently, 
such techniques are frequently not well suited for purification of 
modified oligonucleotides such as methylphosphonates (no charge), 
2'-O-alkyl (hydrophobic) substituted oligonucleotides, and other 
oligonucleotides with modifications that similarly affect the 
oligonucleotides charge and hydrophobicity/hydrophilicity. Prior art 
techniques can also be expensive and time-consuming, particularly in large 
scale operations. Accordingly, new techniques for oligonucleotide 
purification that obviate these problems are desirable. 
SUMMARY OF THE INVENTION 
The present invention provides novel multimeric oligonucleotides and their 
use for the purification of antisense oligonucleotides through 
complementary base recognition and size exclusion principles. In a 
particular embodiment, the invention provides compounds and methods for 
the purification of full length synthetic oligonucleotides from a solution 
also containing N-1 and other failure sequences generated during 
synthesis. The oligonucleotides of the invention are of a unique structure 
that allows them to hybridize specifically with a desired, full-length 
oligonucleotide and concomitantly form multimer aggregates. The desired 
oligonucleotide is thereby bound in a multimer aggregate while other 
undesired oligonucleotides remain in solution. The desired oligonucleotide 
is then separated and isolated using size-exclusion techniques. 
In one aspect of the invention, the oligonucleotides according to the 
invention have at least three structural features: (1) a multimerization 
domain, (2) a hybridization domain, and (3) a linker domain. The 
multimerization domain comprises a sequence of nucleotides that induces 
interoligonucleotide aggregate formation. The hybridization domain 
comprises a sequence of nucleotides that is complementary to the 5' end of 
a synthetic oligonucleotide whose purification is desired. The linker 
domain comprises a sequence of nucleotides or non-nucleotide chemical 
moieties and serves both to link the hybridization domain to the 
multimerization domain as well as space the hybridization domain away from 
the multimerization domain to allow for simultaneous aggregate formation 
and hybridization to the target oligonucleotide. 
When contacted with a crude solution containing the desired oligonucleotide 
and other failure sequences generated during synthesis, the hybridization 
domain hybridizes to the desired ("target") oligonucleotide but not to the 
failure sequences, which are insufficiently complementary to hybridize to 
the hybridization domain under the conditions chosen. The multimerization 
domains concomitantly induce interoligonucleotide aggregation. The target 
oligonucleotide is bound in the resulting aggregate. Failure sequences 
remain free in solution and can be separated from the aggregates using 
standard size exclusion chromatographic techniques. The aggregates bearing 
the target oligonucleotides are then subjected to conditions that cause 
the target oligonucleotide to disassociated from the aggregate, and the 
free target oligonucleotide is isolated from the target-free aggregate by 
size exclusion chromatographic techniques. 
In another embodiment, the present invention provides oligonucleotide 
dendrimers comprised of a branched chemical core structure chemically 
linked to oligonucleotides comprising sequences complementary to one or 
more target oligonucleotides. Oligonucleotide dendrimers according to the 
invention are used in the same way as the multimeric oligonucleotides to 
purify a desired oligonucleotide. 
Use of the present compounds and methods results in superior purity of the 
desired oligonucleotide compared to prior art methods. The present 
invention depends on specific nucleotide base sequence recognition and not 
on charge or hydrophobicity/hydrophilicity. Accordingly, it is useful 
regardless of how the oligonucleotide is modified. A concurrent benefit of 
the present invention is that the multimeric oligonucleotides are 
reuseable. Thus, it is seen that a problem solved by the present invention 
is provision of compounds and methods for the improved purification of 
oligonucleotides. 
The foregoing merely summarizes certain aspects of the present invention 
and is not intended, nor should it be construed, to limit the invention in 
any way. All patents and other publications recited herein are hereby 
incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention provides compounds and methods useful for purifying 
synthetic oligonucleotides. Oligonucleotides are generally synthesized in 
a stepwise manner, adding one nucleotide (or nucleotide dimer or trimer 
blocks) at a time to the 5' end of a nascent, solid-support-bound 
oligonucleotide. Because the reaction that adds each additional nucleotide 
has less than 100% yield, the final oligonucleotide composition is a 
mixture of the desired full length oligonucleotide (of "N" nucleotides in 
length) and failure sequences, which are shorter oligonucleotides, being 
one or more nucleotides shorter in length. These failure sequences are 
often referred to as N-1, N-2, etc., oligonucleotides, the number 
indicating how many fewer nucleotides there are as compared to the full 
length desired oligonucleotide. Prior art techniques for separating the 
full length ("target") oligonucleotide from the failure sequences 
(particularly the N-1 sequence) have met with limited success. The 
compounds and methods of the present invention provide a substantial 
improvement over the prior art. 
The compounds of the invention are oligonucleotides whose structure enables 
them to form oligonucleotide multimer aggregates with the target 
oligonucleotide, but not with the failure sequences. The aggregates 
containing the fall length oligonucleotide can then be separated from the 
unbound failure sequences using standard size exclusion chromatographic 
techniques. The methods of the invention comprise the purification 
procedure using the oligonucleotides of the invention. 
In one aspect of the invention, oligonucleotides according to the invention 
(herein called "multimeric oligonucleotides") have three distinct 
structural domains: i) a multimerization domain, ii) a flexible linker, 
and iii) a hybridization domain. In one embodiment of this aspect of the 
invention, the multimerization domain is a sequence of 4-10 nucleotides 
that, when present in a longer oligonucleotide, results in the 
oligonucleotide aggregating into multimers, hence the name 
"multimerization domain." Nucleotide sequences containing four or more 
contiguous guanine (G) residues form multimeric structures known as G-DNA 
or tetraplex DNA. Sen and Gilbert, Nature 364, 334 (1988). Consequently, 
in one embodiment of the invention, the multimerization domain is a short 
sequence of G nucleotides, generally about 4 to 10 nucleotides. The 
multimerization domain may contain interruptions of the G nucleotide 
sequence by one or two A or U nucleotides, provided that the ability of 
the multimerization domain to induce tetraplex formation is not defeated, 
which generally means that it contains at least 4 contiguous G 
nucleotides. Preferably, from one to all of the G nucleotides is 
2'-substituted with a tetraplex-stablizing substituent. A number of such 
2'-substituents are known to those skilled in the art and include, for 
instance, --N.sub.3, --F, --Cl, and --OR where R is methyl, ethyl, propyl, 
allyl, and methoxyethoxy. Such substitutions increase the stability of the 
interoligonucleotide tetraplex formed by the multimerization domain. 
A nucleotide or non-nucleotide linker joins multimerization and 
hybridization domains and facilitates independent multimerization and 
hybridization events. The linker can be a nucleotide linker of 2 to 10 
nucleotides that are not complementary to the multimerization or 
hybridization domains (to prevent hybridization between the linker and 
either of these domains). The linker can also be a non-nucleotide linker 
of 2 to 15 constituent members such as, but not limited to, ethylene 
glycol, tri(ethylene glycol), tetra(ethylene glycol), penta(ethylene 
glycol) and hexa(ethylene glycol). Other suitable linkers are known in the 
art, some of which are described in U.S. Pat. Nos. 5,321,131, 5,536,821, 
and 5,510,471. 
The hybridization domain consists of a sequence of about 6 to 15 
nucleotides that is complementary in the Watson-Crick sense to one of the 
ends of an antisense oligonucleotide whose purification is desired (e.g., 
for example, oligonucleotide sequence SEQ ID NO 4, infra). Alternatively, 
the hybridization domain may comprise peptide nucleic acids (PNAs) having 
a sequence complementary to the target nucleic acid. See Nielson et al. 
(J. Mol. Recognition 7, 165 (1994)) for a discussion of PNAs. Although we 
generally recite oligonucleotides and nucleic acids in the hybridization 
domain as well as being targets, it will be appreciated by those skilled 
in the art that the hybridization domain, target, or both can be PNAs. 
Alternatively, in this aspect of the invention, the multimeric 
oligonucleotides can comprise a single multimerization domain with a 
linker domain and corresponding hybridization domain on both the 3' and 5' 
end. In this embodiment the multimerization domain can be 4 to 15 
nucleotides long (preferably 10-15) and is further defined as before. The 
linker and hybridization domains are as defined above. The two linkers and 
the two hybridization domains of each multimeric oligonucleotide can be 
the same or different. 
Oligonucleotides according to the invention can be modified in the base, 
sugar, or internucleoside linkage as desired for the particular 
application, e.g., to increase binding affinity. Essentially any 
modification is acceptable so long as it does not prevent the 
oligonucleotide from performing its designated function. A number of such 
modification are known to those skilled in the art. Agrawal and Iyer 
(Current Opin. Biotech. 6, 12 (1995)) review a number of these 
modifications. See also Uhlmann and Peyman (Chem. Rev. 90, 543. (1990)). 
The concept underlying this aspect of the invention is schematically 
displayed in FIG. 2. The multimerization domains of several multimeric 
oligonucleotides become associated through mutual interactions (e.g., 
Hoogsteen base pairing in the case of oligonucleotide (G) multimerization 
domains). This brings several of multimeric oligonucleotides together to 
form an oligonucleotide aggregate (structures A and B of FIG. 2). When a 
solution of synthetic oligonucleotides having a sequence complementary to 
the hybridization domain of the multimeric oligonucleotides is added, the 
synthetic oligonucleotides hybridize to the hybridization domain of the 
multimeric oligonucleotides via Watson-Crick base pairing. Conditions are 
selected so that N-1 and other failure sequences having fewer bases 
complementary to the multimeric oligonucleotides' hybridization domain do 
not hybridize to the multimeric oligonucleotides. The appropriate 
conditions will depend on a number of factors, including target nucleic 
acid length and sequence. It is a routine matter to adjust the 
temperature, salt concentration (ionic strength), and pH of the buffer to 
obtain the desired stringency and, consequently, selectivity. The result 
is that the target oligonucleotide becomes part of a large oligonucleotide 
aggregate (structures C and D in FIG. 2), whereas the synthetic 
oligonucleotide failure sequence remain in solution as individual (i.e., 
unassociated) molecules. The oligonucleotide aggregates containing the 
target oligonucleotide are then separated from the synthetic failure 
sequences using standard size exclusion chromatography (e.g., sephadex or 
sephacryl columns) under conditions selected to maintain the integrity of 
the aggregates. Once again, the conditions (pH, temperature, salt 
concentration) are application dependent but can be adjusted in a routine 
manner to obtain the desired result. A schematic depiction of the process 
is displayed in FIG. 7. 
As can be appreciated from the foregoing, the linker domain serves to 
distance the hybridization domain of the multimeric oligonucleotides from 
the aggregated multimerization domain core of the oligonucleotide, making 
the hybridization domain appear to its binding partner (i.e., the 
complementary target oligonucleotide) like an individual oligonucleotide 
free in solution. Detrimental interactions with the multimer core of the 
aggregate is thereby avoided, facilitating hybridization of the synthetic 
oligonucleotide to the hybridization domain. 
Oligonucleotides according to this aspect of the invention can be 
synthesized using standard techniques. E.g., Methods in Molecular Biology, 
Vol. 20, supra. 
Another aspect of the invention is the aggregates formed by the multimeric 
oligonucleotides. These aggregates generally comprise four or more 
multimeric oligonucleotides that self-associate via the multimerization 
domains. See, e.g., Sen and Gilbert et al. 
In another embodiment, the multimerization domain is a non-nucleotide 
chemical moiety that contains several (about 2 to 20) amino, hydroxyl, or 
carboxyl functional groups (including combinations thereof) that permit 
attachment of hybridization domain(s) (as defined above) to the 
multimerization domain with or without a linker. In this aspect, the 
present invention provides oligonucleotide dendrimers. Oligonucleotide 
dendrimers generally comprise two or three components: a chemical core, 
optionally a linker domain, and from 2 to about 16 hybridization domains. 
The linker domains and the hybridization domains of the oligonucleotide 
dendrimers are the same as defined previously for the multimeric 
oligonucleotides. The oligonucleotides of the oligonucleotide dendrimers 
are linked to the chemical core either directly or through the linker 
domain. Two types of oligonucleotide dendrimers are displayed in FIG. 3. 
As can be appreciated from FIG. 3 and the purpose for which the 
oligonucleotide dendrimers are intended, the oligonucleotides of the 
oligonucleotide dendrimers are preferably linked to the chemical core 
(directly or through a linker) in such a manner as to make them accessible 
in solution for hybridization to the desired oligonucleotide. 
As noted, the oligonucleotides of the oligonucleotide dendrimers have a 
hybridization domain that is complementary to the oligonucleotide to be 
purified or detected. This hybridization domain can comprise the entire 
oligonucleotide or only a portion thereof. In either instance, the 
hybridization domain need only be sufficiently long so as to specifically 
hybridize to the oligonucleotide of interest under acceptable conditions. 
The only other structural constraint on the oligonucleotide dendrimers is 
that they do not have any modifications or other features that would 
interfere with their principal function, i.e., hybridizing with 
complementary oligonucleotides in solution. 
Based upon the foregoing discussion and the theme depicted in FIG. 3, those 
skilled in the art will appreciate that a wide variety of dendrimer 
structures fall within the spirit and scope of the present invention, and 
any such structure can be used in the invention so long as it conforms to 
the requirements set forth herein, including that it is water soluble. A 
number of suitable dendrimeric structures are known. E. g., Newkome et 
al., Aldrichemica Acta 25, 31(1992). 
A preferred oligonucleotide dendrimer of the invention is a branched 
dendrimer, an example of which is shown in FIG. 3A. Such dendrimers are 
synthesized using branching CPG solid support and branching 
phosphoramidites. For instance, such oligonucleotide dendrimers can be 
synthesized on CPG (2000 or 3000 .ANG. pore size) (see Example 1, infra) 
attached to 1,3-didimethoxytrityl-2-glycerol. Symmetric branching 
phosphoramidite for use in the synthesis is commercially available (e.g, 
Clontech, Palo Alto, Calif.). By this method one can control the number of 
branches required to the range of 2-16. 
Another preferred oligonucleotide dendrimer of the invention is a 
STARBURST.TM. dendrimer, as shown in FIG. 3B. STARBURST.TM. dendrimers are 
synthesized by conjugating STARBURST.TM. (PAMAM) dendrimer (Aldrich, 
Milwaukee, Wis.) with oligonucleotide sequences (linker and hybridization 
domains) using the chemistry shown in FIG. 6. Any generation STARBURST.TM. 
dendrimer can be used so long as the oligonucleotide dendrimer is water 
soluble. 
Other dendrimers suitable for use in the present invention include Star 
PEGs and Branched PEGs (PEG=polyethylene glycol), commercially available 
from Shearwater Polymers, Inc. (Huntsville, Ala.). These are multi-armed 
PEGs made by polymerization of ethylene oxide from a cross-linked divinyl 
benzene core. Gnanou et al., Makromol. Chem. 189, 2885 (1988); Rein et 
al., Acta Polymer 44, 225 (1993). Still other dendrimeric molecules useful 
in the invention can be synthesized according to the methods of Rein et 
al. (Acta Polymer 44, 225 (1993)) and references cited therein, and 
Merrill (U.S. Pat. No. 5,171,264), both of which disclose polyethylene 
oxide star molecules. 
Oligonucleotide dendrimers according to the invention can be synthesized 
with other than the STARBURST.TM. dendrimer using the protocol outlined in 
FIG. 6 with only minor modifications. 
In another aspect, the invention comprises methods of separating, 
purifying, and detecting oligonucleotides. In one embodiment of this 
aspect of the invention, the method comprises contacting a solution 
containing an oligonucleotide whose separation, purification, or detection 
is desired with multimeric oligonucleotides or oligonucleotide dendrimers 
of the invention having a hybridization domain sufficiently complementary 
to the desired oligonucleotide to specifically hybridize to the desired 
oligonucleotide (but not to other oligonucleotides having similar 
sequences) under the conditions selected. After hybridization has taken 
place, the solution is subjected to size exclusion chromatography with an 
elution buffer selected to maintain the integrity of the aggregates. The 
elution buffer temperature, pH, and salt concentration are application 
dependent but routine to determine. The fraction containing the multimeric 
oligonucleotide aggregates to which the desired oligonucleotide is bound 
will generally be the fastest moving component on the column. Detection is 
done at standard wavelengths, and the fraction containing the first 
component off the column is collected. The aggregate-containing fraction 
is subject to a buffer that causes the target oligonucleotide to 
disassociate from the multimeric oligonucleotide or oligonucleotide 
dendrimer and then separated by size exclusion chromatography. As before, 
suitable buffer conditions are arrived at through routine adjustment of 
temperature, ionic strength and pH. Two fractions are collected, one 
containing the purified target oligonucleotide and one containing the 
multimeric oligonucleotide or oligonucleotide dendrimer (whichever is 
used). The multimeric oligonucleotide or oligonucleotide dendrimer can 
then be reused for additional purifications. 
As an alternative to size exclusion chromatography, the present methods can 
be conducted with a small pore filter membrane such as micricon and 
centricon filters available from Amicon (Beverly, Mass.), which are 
particularly useful for small scale purification like those in which PCR 
is used. Membrane filter cartridges or pumps with suitable pore sizes can 
be used for large scale purification. Such filter membranes of course will 
have a pore size chosen to allow unbound failure sequences to pass through 
but not the multimeric oligonucleotide (or oligonucleotide 
dendrimer)/target nucleic acid aggregate. In this aspect of the invention, 
the solution containing the aggregate and failure sequences is filtered 
through the membrane. As before, the solution buffer is chosen to be of 
such stringency as to allow selective hybridization of the target sequence 
but not other oligonucleotides to the hybridization domain. The filtrate 
(containing the failure sequences) can be discarded if desired. The 
material remaining on the filter, which is the aggregate, is then washed 
in situ with a buffer having a stringency that causes the target to 
disassociate from the oligonucleotide multimer or dendrimer 
oligonucleotide. The unbound target oligonucleotide will pass through the 
membrane. The filtrate containing the isolated oligonucleotide is 
collected. The membrane, on which the oligonucleotide multimer or 
dendrimer oligonucleotide is, can be washed and the oligonucleotide 
multimer or dendrimer oligonucleotide collected and used again. 
The present compounds and methods can also be used to isolated segments 
from a genome. In this aspect of the invention the hybridization domain of 
the multimeric oligonucleotide or oligonucleotide dendrimer is 
complementary in the Watson-Crick, Hoogsteen, reverse Watson-Crick, or 
reverse Hoogsteen sense. The genome or portion thereof containing the 
segment of interest (the target segment) is subject to restriction 
endonucleases and the resulting hydrolysate subject to the methods of the 
invention. If the target segment is purine rich, the hybridization domain 
of the multimeric oligonucleotide or oligonucleotide dendrimer may be 
complementary in the Hoogsteen or reverse Hoogsteen sense to the purine 
rich segment so that the double stranded target segment hybridizes to the 
hybridization domain to form a triplex. Alternatively, where the target 
segment is a mixed sequence of purine and pyrimidines, it is preferably 
denatured first and the hybridization domain is complementary in the 
Watson-Crick sense to one of the strands. 
It will be appreciated from the foregoing that the compounds and methods of 
the invention provide several advantages over prior art techniques. The 
present methods can be used to purify any molecule capable of specific 
hybridization based on complementary nucleotide base sequence recognition 
through Watson-Crick, reverse Watson-Crick, Hoogsteen, or reverse 
Hoogsteen hydrogen bonding, including but not limited to nucleic acids and 
PNAs. Most notably, the methods of the invention do not rely on charge, 
hydrophobicity, or hydrophilicity and, therefore, are suitable regardless 
of how the target oligonucleotide (or PNA) is modified. An additional 
advantage of the methods of the invention is that the multimeric 
oligonucleotide can be used repeatedly for a number of purifications, 
thereby reducing the cost. The results presented herein demonstrate that 
the multimeric structures can be used to purify antisense oligonucleotides 
as shown in FIG. 7. 
From the foregoing, those skilled in the art will appreciate that the 
compounds and methods of the invention are not limited to isolating a 
single synthetic oligonucleotide from a mixture of failure sequences, but 
can be used to isolate a desired oligonucleotide from any composition in 
solution. Furthermore, by using compounds of the invention with two or 
more different hybridization domains or mixtures of the compounds of the 
invention in which each compound has one of several different 
hybridization domains, more than one target oligonucleotide can be 
separated at a time. In this embodiment, multimeric oligonucleotides or 
asymmetric branching dendrimers (commercially available from, for example, 
Clontech (Palo Alto, Calif.)) are used. 
The following examples are provided for illustrative purposes only and are 
not intended, nor should they be construed, to limit the invention in any 
way. Those skilled in the art will appreciate that variations can be made 
without violating the scope or spirit of the invention. 
EXAMPLES 
FIG. 1 displays the oligonucleotide sequences and aggregates used in this 
study. The three boxes (from left to right) in each of the first three 
structures represent multimeric, linker, and hybridization domains, 
respectively, as described herein. In dendrimer 2, the number of branches 
is equal to 2n, and where n is 2-4. In dendrimer 3, the number of branches 
is equal to n', and where n' is 2-16 (n'=12 is shown in FIG. 3). 
Oligonucleotide SEQ ED NO 4 is the antisense oligonucleotide whose 
purification is undertaken, and the underlined portion is complementary to 
the hybridization domains of oligonucleotide SEQ ID NO 1 and dendrimers 2 
and 3. 
EXAMPLE 1 
Synthetic Methods 
Oligonucleotide Synthesis 
Oligonucleotides were synthesized on a Milligen 8700 DNA synthesizer 
(Bedford, Mass.) using phosphoramidite chemistry. 
P-cyanoethyl-N,N-diisopropyl phosphoramidites were purchased from 
Millipore for DNA synthesis. Oligonucleotide SEQ ID NO 2 was synthesized 
on CPG (2000 or 3000 .ANG. pore size) attached to 
1,3-didimethoxytrityl-2-glycerol. Symmetric branching phosphoramidite was 
obtained from Clontech (Palo Alto, Calif.). After attaching the required 
number of branches on the synthesizer, oligonucleotide synthesis was 
continued with normal P-cyanoethyl-N,N-diisopropyl phosphoramidites as 
described above. 
After synthesis, oligonucleotides SEQ ID NOs 1 and 2 were deprotected, 
purified on reverse phase HPLC (C.sub.18), detritylated, and desalted 
using C.sub.18 Sep-pack cartridges (Waters, Milford, Mass.). 
Oligonucleotide SEQ ID NO 4 was deprotected and used as is without 
purification. 
Synthesis of Branching Dendrimer 
We synthesized 1,3-di-dimethoxytrityl-2-hydroxy-glycerol by treating 
glycerol with 2 equivalents of 1,1'-dimethoxytrityl chloride (DMTCl) in 
pyridine for 24 hrs at room temperature and for additional 7 hrs at 
50.degree. C. to allow completion of the second hydroxy group with DMTCl. 
The di-DMT-2-hydroxy glycerol was purified by flash column chromatography 
on silica gel 60 (&lt;230 mesh ASTM, Merck, Darmstadt, Germany) by eluting 
with a mixture of hexane:methylene chloride:triethylamine (20:4:1). Yield 
of the purified white diDMT product was 94%. 
The diDMT product obtained was loaded on to long chain alkylamido propionic 
acid controlled-pore glass (CPG) supports (CPG, Inc., Lincoln Port) having 
pore sizes of 2000 and 3000 .ANG. as reported (Damha et al., Nucleic Acids 
Res. 18, 3813 (1990)) to yield 1,3-diDMT-2-hydroxy-glycerol on CPG. The 
loading efficiencies were 13.8 .mu.moles/gm (2000 .ANG.) and 9.3 .mu.moles 
gm (3000 .ANG.). 
Conjugation of Oligonucleotide to STARBURST.TM. Dendrimer 
The 3 '-DMT-5'-P-cyanoethyl-N,N-diisopropyl phosphoramidites and 5'-monomer 
attached CPG solid support were obtained from Glen Research Laboratories 
(Sterling, Va.) or Chemgenes (Waltham, Mass.). Dendrimer 5 (FIG. 6) was 
synthesized on a 5'-monomer attached CPG using 3'-DMT-5'-phosphoramidite 
monomers with longer detritylation coupling times and in trityl-off mode. 
Then the CPG-attached oligonucleotide with free 3'-OH was activated with 
bis(p-nitrophenyl) carbonate in anhydrous 1,4-dioxane with triethylamine 
as catalyst (3 drops) for 1 hr to obtain active carbonate dendrimer 6 
(FIG. 6) as described by Habus et al. (Bioorganic and Med Chem. Lett. 4, 
1065 (1994)) and Habus et al. (Bioconjugate Chem. 6, 327 (1995)). The 
active oligonucleotide carbonate was then successively washed with 
anhydrous 1,4-dioxane and acetonitrile and dried in vacuum. The activated 
oligonucleotide was taken in anhydrous pyridine/methanol and added to 
STARBURST.TM. (PAMAM) dendrimer, generation 2 (Mol. Wt. 3,256) (Aldrich 
Chemical Co. Milwaukee) and shaken in a mechanical shaker for 2 hrs at 
room temperature to give dendrimer 7 (FIG. 6). Then the product was washed 
with pyridine and methanol. Dendrimer 3 (FIG. 1) was cleaved from CPG and 
deprotected as described above. Then the aggregate was purified on 
polyacrylamide gel. 
EXAMPLE 2 
Purification With Multimeric Oligonucleotides 
Crude oligonucleotide SEQ ID NO 4 was labeled at the 5'-end with .sup.32 P 
using T4-polynucleotide kinase (Promega, Madison, Wis.), and 
.gamma.-.sup.32 P-dATP (Sambrook et al., Molecular Cloning (Cold Spring 
Harbor Press, Cold Spring Harbor, N.Y., 1989.)). Tetraplex (multimeric 
structure) formation was confirmed on gel using 5'-end .sup.32 P labeled 
oligonucleotide SEQ ID NO 1 in 200 mM potassium acetate. For purification 
experiments, the required amount of labeled, cold oligonucleotide SEQ ID 
NO 4 was mixed with oligonucleotides SEQ ID NO 1 in 10 mM sodium 
dihydrogen phosphate (pH 7.6) and 100 mM sodium chloride and incubated for 
30-60 min. Then the samples were loaded on 20% non-denaturing 
polyacrylamide gel and electrophoresed. The slow moving band was excised 
from the gel and eluted with 0.5 mM sodium acetate (pH 7.2) overnight and 
ethanol precipitated. The DNA sample was then mixed with formamide gel 
loading buffer and analyzed on a denaturing 7M urea polyacrylamide gel. 
FIG. 4A is an autoradiogram of non-denaturing polyacrylamide gel showing 
multimeric structures formed with oligonucleotide SEQ ID NO 1. Lanes 1 and 
2 are markers of 22-base long single- and double-stranded DNAs, 
respectively. Lanes 3-6 contain oligonucleotide SEQ ID NO 1 incubated in 
100 mM NaOAc, pH 7.6 (lane 3), 200 mM NaOAc, pH 7.6 (lane 4), 100 mM KOAc, 
pH 7.6 (lane 5), and 200 mM KOAc, pH 7.6 (lane 6). The figure shows that 
SEQ ID NO 1 forms multimeric structure (tetraplex) as expected. 
FIG. 4B is an autoradiogram showing homogeneity of SEQ ID NO 4 purified 
with multimeric oligonucleotide SEQ ID NO 1. Lanes 5 and 6 are crude SEQ 
ID NO 4 before and after treating with SEQ ID NO 1. Lane 1 is purified SEQ 
ID NO 4 by standard methods. Lanes 2-4 are SEQ ID NO 4 prepurified by 
conventional techniques and subsequently treated with SEQ ID NO 1 as in 
the case of crude SEQ ID NO 4. This figure shows that the isolated 
fraction contains only pure antisense oligonucleotide SEQ ID NO 1 (lanes 
2, 3, 4, and 6) (compare with lanes 1 and 5, which contain purified and 
crude oligonucleotide SEQ ID NO 4, respectively). 
EXAMPLE 3 
Purification With Oligonucleotides Dendrimers 
We used oligonucleotide dendrimers shown in FIG. 3A to demonstrate their 
use for purification of antisense oligonucleotide SEQ ID NO 4. Dendrimer 2 
(FIG. 1) was used for purification in the same manner as SEQ ID NO 1, as 
described in Example 2. 
FIG. 5A is an autoradiogram of a non-denaturing gel showing the mobility of 
the complexes of crude SEQ ID NO 4 with 2 and 4 branched dendrimer 2 (FIG. 
1) (lanes 3 and 4, respectively). Lane 1 is crude SEQ ID NO 4 alone. Lane 
2 is crude SEQ ID NO 4 with a 25 base long complementary sequence. Note 
that the failure sequences are seen as a diffuse band below the dark band 
in lane 1, not seen in lane 2, but present in lanes 3 and 4. Appropriate 
bands were cut, eluted, and run on a denaturing polyacrylamide gel to 
examine the homogeneity of oligonucleotide SEQ ID NO 4. FIG. 5B is an 
autoradiogran of a denaturing gel showing homogeneity of the bands excised 
from lanes 2 (lane 1), 3 (lane 2) and 4 (lane 3) of the gel shown in FIG. 
5A. Lanes 2 and 3 of FIG. 5B suggest that the oligonucleotide is pure and 
the failure sequences are absent. Similar results obtained with the 
oligonucleotide dendrimer shown in FIG. 3B. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 4 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 22 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
# 22GGG GG 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 13 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 13 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 14 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 14 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 25 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: other nucleic acid 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: YES 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 25 TCTC CTTCT 
__________________________________________________________________________