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(12) Patent Application Publication (10) Pub. No.: US 2011/0160082 A1 WOO Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2011/0160082 A1 WOO Et Al US 2011 0160O82A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0160082 A1 WOO et al. (43) Pub. Date: Jun. 30, 2011 (54) MOBILITY-MODIFIED NUCLEOBASE Publication Classification POLYMERS AND METHODS OF USING (51) Int. Cl. SAME C40B 30/04 (2006.01) C7H 2L/00 (2006.01) C7H 2L/02 (2006.01) (75) Inventors: Sam L. WOO, Redwood City, CA C7H 2L/04 (2006.01) (US); Ron GRAHAM, Carlsbad, C40B 40/06 (2006.01) CA (US); Jing TIAN, Mountain C07F 9/24 (2006.01) View, CA (US) (52) U.S. Cl. ............... 506/9:536/23.1; 506/16; 558/185 (57) ABSTRACT (73) Assignee: LIFE TECHNOLOGIES CORPORATION, Carlsbad, CA The present invention relates generally to nucleobase poly merfunctionalizing reagents, to mobility-modified sequence (US) specific nucleobase polymers, to compositions comprising a plurality of mobility-modified sequence-specific nucleobase (21) Appl. No.: 13/009,827 polymers, and to the use of Such polymers and compositions in a variety of assays, such as, for example, for the detection of a plurality of selected nucleotide sequences within one or (22) Filed: Jan. 19, 2011 more target nucleic acids. The mobility-modifying polymers of the present invention include phosphoramidite reagents Related U.S. Application Data which can be joined to other mobility-modifying monomers and to sequence-specific oligonucleobase polymers via (60) Continuation of application No. 1 1/456,836, filed on uncharged phosphate triester linkages. Addition of the mobil Jul. 11, 2006, now Pat. No. 7,897,338, which is a ity-modifying phosphoramidite reagents of the present inven division of application No. 10/856,754, filed on Jun. 1, tion to oligonucleobase polymers results in unexpectedly 2004, now Pat. No. 7,235,927, which is a division of large effects the mobility of those modified oligonucleobase application No. 09/836,704, filed on Apr. 16, 2001, polymers, especially upon capillary electrophoresis in non now Pat. No. 6,743,905. sieving media. US 2011/0160082 A1 Jun. 30, 2011 MOBILITY-MODIFIED NUCLEOBASE In the illustration, DMT represents dimethoxytrity1 and iPr POLYMERS AND METHODS OF USING represents isopropyl. When x=5, each PEO reagent added to SAME the nucleobase polymer imparts the nucleobase polymer with an electrophoretic retardation of approximately 2 nucleotides 1. CROSS REFERENCE OF RELATED as compared to the unmodified nucleobase polymer under APPLICATIONS both sieving and nonsieving electrophoretic conditions. Due to limitations of DNA chemistry, no more than about 40 PEO 0001. This application is a divisional of pending U.S. reagents can be coupled to a nucleobase polymer and result in application Ser. No. 10/856,752, filed May 28, 2004, which is homogenous product. Accordingly, the greatest electro a divisional of U.S. application Ser. No. 09/836,704, filed phoretic mobility retardation that can be achieved using these Apr. 16, 2001 (now U.S. Pat. No. 6,743,905), all of which are standard PEO modifying reagents is about 80 nucleotides. incorporated herein by reference. 0006. However, in light of the increasing need to simulta neously analyze vast numbers of nucleotide sequences in a 2. FIELD OF THE INVENTION single experiment, e.g., the 200 identified alleles associated 0002 The present invention relates generally to nucleo with cystic fibrosis, there remains a need in the art for new base polymer functionalizing reagents, to mobility-modified mobility-modifying polymers that have different charge to sequence-specific nucleobase polymers, to compositions translational frictional drag ratios than currently available comprising a plurality of mobility-modified sequence-spe mobility-modifying polymers, and that can impart electro phoretic mobility retardations of greater than the 80 nucle cific nucleobase polymers, and to the use of Such polymers otides achievable with available PEO modifying reagents. and compositions in a variety of assays, such as, for example, The availability of such new mobility-modifying polymers for the detection of a plurality of selected nucleotide would greatly increase the repertoire of available mobility sequences within one or more target nucleic acids. modifications, thereby enabling the ability to perform 3. BACKGROUND OF THE INVENTION extremely complex sequence analyses in simple, preferably automated, formats. 0003 Methods used to detect selected nucleotide sequences within target nucleic acids underlie an extensive 4. SUMMARY OF THE INVENTION array of practical applications including, but not limited to, 0007. In one aspect, the present invention provides mobil paternity testing, forensic analysis, organ donor recipient ity-modifying phosphoramidite functionalizing reagents matching, disease diagnosis, prognosis and treatment, and comprising a polymeric portion and a phosphoramidite moi prenatal counseling. ety. The phosphoramidite moiety comprises an oxygen-pro 0004. There exists a need in the art for materials and meth tecting group that, quite unlike the B-cyanoethyl oxygen pro ods that permit pluralities of selected nucleotide sequences to tecting group used in conventional phosphoramidite reagents, be simultaneously detected and analyzed, under uniform is stable to basic conditions such as the conditions and experimental conditions, preferably in a single, automated, reagents used in conventional phosphoramidite oligonucle assay reaction. One approach towards meeting this need has otide synthesis and deprotection. As a consequence of this been the development of mobility-modifying polymers that stable oxygen protecting group, the bond formed between the can be attached to sequence-specific nucleobase polymers functionalizing reagent and the compound functionalized is that act to increase the effective size of the modified nucleo an uncharged phosphate triester. The mobility-modifying base polymers. Where the charge to translational frictional phosphoramidite reagents of the invention may be used to draw ratio of the mobility-modifying polymer differs from functionalize a wide variety of Substances and materials to that of the nucleobase polymer to which it is attached, the add mass and size to the Substance or material without Sub resulting modified nucleobase polymer will have an electro stantially altering its overall net charge. phoretic mobility that differs from that of the unmodified 0008. The mobility-modifying phosphoramidite reagents nucleobase polymer. This alteration of the charge to transla of the invention are compatible with standard phosphoramid tional frictional drag ratio may be employed in various appli ite synthetic schemes and can be used with commercially cations to effect electrophoretic separation of similarly-sized available nucleobase polymer synthesis instruments. Thus, nucleobase polymers under both sieving and non-sieving the mobility-modifying phosphoramidite reagents of the conditions. present invention are particularly convenient for mobility 0005. The most commonly employed mobility-modifying modifying synthetic sequence-specific nucleobase polymers polymers are polyethylene oxides (PEO) that are attached to Such as, for example, 2'-deoxyoligonucleotides. They may be a nucleobase polymer using standard DNA chemistry (Gross readily attached to the 5'-terminus of the nucleobase polymer, man et al. (1994) Nucleic Acids Research 22 (21): 4527–34). to the 3'-terminus, or to both the 5’- and 3'-termini, depending An exemplary standard PEO phosphoramidite reagent (“PEO on the particular application and/or desired degree of mobil reagent’) that can be added to a nucleobase polymer using ity modification. standard DNA chemistry is illustrated below: 0009. The terminus of the polymeric portion that is distal to the phosphoramidite moiety may be protected with a group that is selectively removable under the desired synthesis con 1N1 CN ditions, or it may comprise a group that is essentially non reactive, Such as, for example an alkyl, aryl, arylakyl, etc. pro-ha-'l-no o K group. In the former embodiment, the protecting group may N--iPr) be selectively removed for sequential condensation of one or more additional phosphoramidite reagents. Suitable selec tively removable protecting groups will depend upon the US 2011/0160082 A1 Jun. 30, 2011 identity the group being protected and will be apparent to the invention permit the formation of mobility-modified sub those of skill in the art. Selectively removable groups suitable stances comprising branched or dendritic polymer segments. for protecting hydroxyl groups include, by way of example 0013. In one convenient embodiment, the polymer portion and not limitation, any of the acid-labile groups that are of the mobility-modifying phosphoramidite reagents of the commonly used to protect the 5'-hydroxyl of conventional nucleoside phosphoramidites oligonucleotide synthesis present invention is a polyalkylene oxide, an illustrative reagents. Such as acid-labile trityl groups (e.g., monomethox embodiment of which is depicted as Formula (I) below: ytrityl, dimethoxytrityl, etc.). 0010. The mobility-modifying phosphoramidite reagents of the invention may be used alone to mobility-modify sub stances such as nucleobase polymers to add mass and size to the nucleobase polymer without altering its overall net charge. Alternatively, they may be used in conjunction with conventional mobility-modifying reagents, such as the PEO reagent illustrated above, to mobility-modify Substances Such as nucleobase polymers. Because the mobility-modifying 0014 wherein: phosphoramidite reagents of the invention may be used to add
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