Taxol resistance mediated by transfection of the liver-specific sister gene of P-glycoprotein

The synthesis and properties of novel RNA mimetics, oligoribonucleotide N3'-->P5' phosphoramidates, are described. These oligonucleotides contain 3'-aminoribonucleosides connected via N3'-->P5' phosphoramidate linkages, replacing the native RNA O3'-->P5' phosphodiester counterparts. The key monomers 2'-t-butyldimethylsilyl-3'-(monomethoxytrityl)-amino-5'-phospho ramidi tes were synthesized and used to prepare the oligonucleotide phosphoramidates using a solid phase methodology based on the phosphoramidite transfer reaction. Oligoribophosphoramidates are very resistant to enzymatic hydrolysis by snake venom phosphodiesterase. These compounds form stable duplexes with complementary natural phosphodiester DNA and RNA strands, as well as with 2'-deoxy N3'-->P5' phosphoramidates. The increase in melting temperature, Delta T m, was 5-14 degrees C relative to the 2'-deoxy phosphoramidates for decanucleotides. Also, the thermal stability of the ribophosphoramidatehomoduplex was noticeably higher (Delta T m +9.5 degrees C) than that for the isosequential 2'-deoxy phosphoramidate complex. Furthermore, the oligopyrimidine ribo N3'-->P5' phosphoramidate formed an extremely stable triplex with an oligopurine/oligopyrimidine DNA duplex with Delta T m +14.3 degrees C relative to the 2'-deoxy N3'-->P5' phosphoramidate counterpart. The properties of the oligoribonucleotide N3'-->P5' phosphoramidates indicate that these compounds can be used as hydrolytically stable structural and functional RNA mimetics.     

Analysis of an oligonucleotide N3'-->P5' phosphoramidate/phosphorothioate chimera with capillary gel electrophoresis

N3'-->P5' phosphoramidate/phosphorothioate chimeric oligonucleotides (ODNs) are presently under investigation as potential antisense drugs. Within the field of antisense research, "second generation" chimeric ODNs have exhibited improved characteristics relative to oligonucleotides with uniformly modified backbones. The ODN of interest for this study consisted of a chemically synthesized 18-mer of mixed nucleotide base sequence with a backbone consisting of eight central phosphorothioate linkages flanked by four N3'-->P5' phosphoramidate (amidate) linkages on the 5'-end and five amidate linkages ont he 3'-end. This chimera presents analytical challenges due to the central phosphorothioate region. Here, we present a capillary gel electrophoresis (CGE) method for the analysis of the above N3'-->P5' phosphoramidate/phosphorothioate chimera. CGE was used to analyze the product prior to its purification by reversed phase - high performance liquid chromatography (RP-HPLC), and each fraction collected from the purification was similarly analyzed. An internal standard was utilized to determine the relative mobility of our product, and polyacrylamide gel electrophoresis (PAGE) analysis was used to verify CGE results.     

alpha-Oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and duplex formation

The synthesis and hybridization properties of novel nucleic acid analogs, alpha-anomeric oligodeoxyribonucleotide N3'-->P5' phosphoramidates, are described. The alpha-3'-aminonucleoside building blocks used for oligonucleotide synthesis were synthesized from 3'-azido-3'-deoxythymidine or 3'-azido-2',3'-dideoxyuridine via acid catalyzed anomerization or transglycosylation reactions. The base-protected alpha-5'-O-DMT-3'-aminonucleosides were assembled into dimers and oligonucleotides on a solid support using the oxidative phosphorylation method.1H NMR analysis of the alpha-N3'-->P5' phosphoramidate dimer structures indicates significant differences in the sugar puckering of these compounds relative to the beta-N3'-->P5' phosphoramidates and to the alpha-phosphodiester counterparts. Additionally, the ability of the alpha-oligonucleotide N3'-->P5' phosphoramidates to form duplexes was studied using thermal denaturation experiments. Thus the N3'-->P5' phosphoramidate decamer containing only alpha-thymidine residues did not bind to poly(A) and exhibited lower duplex thermal stability with poly(dA) than that for the corresponding beta-anomeric phosphoramidate counterpart. A mixed base decamer alpha-CTTCTTCCTT formed duplexes with the RNA and DNA complementary strands only in a parallel orientation. Melting temperatures of these complexes were significantly lower, by 34-47 or 15-25 degrees C, than for the duplexes formed by the isosequential beta-phosphoramidates in antiparallel and parallel orientations respectively. In contrast, the alpha-decaadenylic N3'-->P5' phosphoramidate formed duplexes with both RNA and DNA complementary strands with a stability similar to that of the corresponding beta-anomeric phosphoramidate. Moreover, the self-complementary oligonucleotide alpha-ATATATATAT did not form an alpha:alpha homoduplex. These results demonstrate the effects of 3'-aminonucleoside anomeric configuration on sugar puckering and consequently on stability of the duplexes.     

Synthesis and characterization of a new 5-thiol-protected deoxyuridine phosphoramidite for site-specific modification of DNA

A new nucleotide analogue was developed for site-specific incorporation of a reactive thiol group into DNA. This creates a unique site for the post-synthetic modification of that nucleotide with a variety of molecular tags, such as photo-cross-linkers and fluorescent or spin-label moieties. 5'-O-(4,4'-Dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)thio]-2'-deoxyuridin e 3'-O-(2-cyanoethyl N,N'-diisopropylphosphoramidite) was synthesized and incorporated at internal positions in several oligonucleotides using automated DNA synthesis and standard phosphoramidite chemistry. The coupling yield of the analogue was comparable to the coupling yield for a standard phosphoramidite, and no significant differences were observed in the overall yields of the dinitrophenyl-labeled oligonucleotides compared to the corresponding unmodified oligonucleotides. Characterization of the dinitrophenyl-modified oligonucleotides included enzymatic degradation, HPLC chromatography, and gel electrophoresis. Deprotection of the mercaptan group with beta-mercaptoethanol yielded an oligonucleotide containing 5-mercaptodeoxyuridine which was then selectively modified, without purification, by reaction with 5-(iodoacetamido)fluorescein. Incorporation of the dinitrophenyl-modified oligonucleotide into double-stranded DNA was achieved using the polymerase chain reaction. CHaracterization of the dinitrophenyl-labeled product by immunodetection with anti-dinitrophenyl antibodies confirmed the stability of the protecting group to the thermocycling and thus established the use of this thiol-protected mercaptodeoxyuridine phosphoramidite for preparation of site-specifically modified DNA.     

Comparative sequence analysis of ribonucleases HII, III, II PH and D

Escherichia coli ribonucleases (RNases) HII, III, II, PH and D have been used to characterise new and known viral, bacterial, archaeal and eucaryotic sequences similar to these endo- (HII and III) and exoribonucleases (II, PH and D). Statistical models, hidden Markov models (HMMs), were created for the RNase HII, III, II and PH and D families as well as a double-stranded RNA binding domain present in RNase III. Results suggest that the RNase D family, which includes Werner syndrome protein and the 100 kDa antigenic component of the human polymyositis scleroderma (PMSCL) autoantigen, is a 3'-->5' exoribonuclease structurally and functionally related to the 3'-->5' exodeoxyribonuclease domain of DNA polymerases. Polynucleotide phosphorylases and the RNase PH family, which includes the 75 kDa PMSCL autoantigen, possess a common domain suggesting similar structures and mechanisms of action for these 3'-->5' phosphorolytic enzymes. Examination of HMM-generated multiple sequences alignments for each family suggest amino acids that may be important for their structure, substrate binding and/or catalysis.     

A report card to the nation on adolescents and sexually transmitted diseases

Photolysis of thymidylyl-(3'-->5')-thymidine (TpT) has been previously reported by many workers to lead to only two cyclobutane dimers, the cis-syn and trans-syn-I dimers. This is curious in light of the fact that photolysis of thymidylyl-(3'-->5')-deoxyuridine (TpdU), which has a hydrogen in place of a methyl group at C6 of the 3'-thymidine, produces two trans-syn diastereomers. Recently, we discovered by way of X-ray crystallography that photolysis of the (Rp)-methyl phosphate ester of TpT leads to two trans-syn diastereomers, prompting us to reexamine the photochemistry of TpT. In this paper we show that sensitized photolysis of TpT also leads to the hitherto unknown trans-syn-II diastereomer in 2% yield. We also report the solution-state 1H NMR assignment of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester by way of 2D homonuclear Hartmann--Hahn experiment, rotating frame nuclear Overhauser effect spectroscopy, and proton-detected 1H-31P correlation spectroscopy. Conformational analysis of three-bond 1H-1H, 1H-31P, and 13C-31P coupling constant data established a close similarity between the solution-state structures of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester, with the crystal structure of the methyl phosphate ester.     

Effect of Hg(II) on the spectroscopic properties of DNA bases: circular dichroism of deoxyadenosine and thymidine monomers and dimers

The addition of Hg(ClO4)2(Hg(II)) to 2'-deoxyadenosine (dA), thymidine (dT), to their respective 5'-monophosphates (dAp,dTp) as well as to the dinucleoside phosphates 2'-deoxyadenylyl-(3'-->5')-2'-deoxyadenosine (d(ApA)), 2-deoxyadenylyl-(3'-->5')thymidine(d(ApT)), thymidylyl-(3'-->5')-2'-deoxyadenosine (d(TpA)), and thymidylyl-(3'-->5')thymidine (d(TpT))--all dissolved in 0.1 M NaClO4, 5 mM cacodylic acid buffer, pH 7--produces major alterations in the circular dichroism (CD) of the dimers but no or only small changes in the CD of the monomers. Of particular interest are the Hg(II)-induced changes in the CD of d(ApT) and d(TpA): they are strongly sequence-dependent and, within reason, progress in a "mirror"-like fashion when the concentration of Hg(II) is varied. In the absence of Hg(II), the CD of the dimers is conservative (d(TpT)), or near-conservative (d(ApA), d(ApT), d(TpA)), but becomes nonconservative upon the addition of Hg(II). The rotational strength R of the various Cotton effects of the dimers was evaluated as a function of Hg(II) concentration. Features of the CD spectra of mercurated d(ApA) and d(TpT) persist in the CD spectra of mercurated poly[d(A).(T)], but there is little obvious agreement of the CD spectra of mercurated d(ApT) and d(TpA) with the CD of mercurated poly[d(A-T).d(A-T)].     

Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog

FEN-1 proteins are a family of nucleases essential for lagging strand DNA synthesis. A gene with sequence similarity to FEN-1 protein-encoding genes, rad2 +, has been identified in Schizosaccharomyces pombe . We report the overexpression, purification, and character-ization of the putative S.pombe FEN-1 homolog, Rad2p. A GST-Rad2p fusion protein was over-expressed in Saccharomyces cerevisiae and purified to near homogeneity by GST affinity chromatography. Although Rad2p had been previously classified as a putative FEN-1 protein based on amino acid homology, there has been no biochemical evidence demonstrating flap endonuclease activity. DNA cleavage analysis of several different oligodeoxynucleotide structuresindicates that GST-Rad2p possesses both 5'-flap endonuclease and 5'-->3' double-stranded DNA exo-nuclease activities. GST-Rad2p incises a 5'-flap and a 5'-pseudo-Y structure one base 3' of the branch point in the duplex region and also degrades double-stranded DNA. This is the first report on the biochemical characterization of S.pombe Rad2p. The potential roles of Rad2p in DNA excision repair and other nucleic acid reactions are discussed.     

A single cleavage assay for T5 5'-->3' exonuclease: determination of the catalytic parameters forwild-type and mutant proteins

Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single cleavage assay for this enzyme which uses a 5'-overhanging hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.     

Exonuclease IX of Escherichia coli removes 3' phosphoglycolate end groups from DNA

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA, and in both a 5'--> 3' and a 3' --> 5' direction. These enzymes are involved in replicative, repair and recombination functions. A new exonuclease recently identified in E. coli, termed exonuclease IX, acts preferentially on single-stranded DNA as a 3'--> 5' exonuclease and also functions as a 3' phosphodiesterase on DNA containing 3' incised apurinic/apyrimidinic (AP) sites to remove the product trans-4-hydroxy-2-pentenal-5-phosphate. We now demonstrate that the enzyme is also able to remove 3' phosphoglycolate end groups from DNA. This activity may have an important role in DNA base excision repair in E. coli.     

DNA cleavage and degradation by the SbcCD protein complex from Escherichia coli

The SbcCD protein is a member of a group of nucleases found in bacteriophage T4 and T5, eubacteria, archaebacteria, yeast, Drosophila, mouse and man. Evidence from electron microscopy has revealed a distinctive structure consisting of two globular domains linked by a long region of coiled coil, similar to that predicted for the members of the SMC family. That a nuclease should have such an unusual structure suggests that its mode of action may be complex. Here we show that the protein degrades duplex DNA in a 3'-->5' direction. This degradation releases products half the length of the original duplex suggesting simultaneous degradation from two duplex ends. This may provide a link to the unusual structure of the protein since our data are consistent with recognition and cleavage of DNA ends followed by 3'-->5' nicking by two nucleolytic centres within a single nuclease molecule that releases a half length limit product. We also show that cleavage is not simply at the point of a single-strand/double-stand transition and that despite the dominant 3'-->5' polarity of degradation, a 5' single-strand can be cleaved when attached to duplex DNA. The implications of this mechanism for the processing of hairpins formed during DNA replication are discussed.     

The detection of oligonucleotide N3'-->P5' phosphoramidate/RNA duplexes with capillary gel electrophoresis

Oligonucleotide N3'-->P5' phosphoramidates (3'-phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single-stranded RNA fragments utilizing 3'-phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3'-Phosphoramidate 9-mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3'-phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3'-phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utilized to verify duplex formation.     

Combining the preparation of oligonucleotide arrays and synthesis of high-quality primers

Based on the oligomer-chip technology, oligonucleotide arrays were synthesized directly on polypropylene sheets by a modified phosphoramidite chemistry using beta-eliminating nucleobase-protecting groups in combination with a succinate solid-phase linker. This method decouples the oligonucleotide deprotection from the support cleavage procedure, in contrast to standard phosphoramidite chemistry. In addition to being reliable substrates for hybridization experiments, the arrays also serve as source for the isolation of individual oligonucleotides. Technically, this allowed for a direct control of the quality of the arrayed oligomers. The released compounds were sufficient in amount and purity to work without further purification in PCR and DNA-sequencing reactions, with the results being identical to controls with commercially obtained primer molecules. Consequences for oligomer-chip hybridization procedures, the applicability of such hybrid-function arrays in, for example, diagnostics or comparative biology, and developments toward parallel primer synthesis are discussed.     

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.     

Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2',5'-ADP-Sepharose, which results in a high yield of pure enzyme with a specific activity at 30 degreesC and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes from Xanthomonas translucens and Escherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, the Pseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol-1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 +/- 0.1 and 9. 7 +/- 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40 degreesC, but activity is regained when the heated enzyme is cooled to 30 degreesC or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

Synthesis and cleavage of oligodeoxynucleotides containing a 5-hydroxyuracil residue at a defined site

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.     

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes

Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.     

Influence of antithyroid antibodies in euthyroid women on in vitro fertilization-embryo transfer outcome

The direct synthesis and subsequent rapid characterisation of multiple antigen peptides (MAPs) for use as immunogens has presented difficulties, partly because of the formation of incomplete or truncated peptide sequences during the synthetic procedure. Therefore, many researchers have resorted to ligation procedures for the synthesis of MAP constructs. This article describes a method to improve the yield of MAP constructs by direct synthesis methods, as well as a general procedure that enables easier characterisation of the synthetic products. In particular, during the synthesis of MAP constructs, a capping procedure was introduced after each amino acid coupling step, thus improving significantly the yield of the desired multi-dendritic peptidic immunogens. Through the use of this capping procedure, problems arising from the incomplete amino acid residue coupling at the point of synthesis were minimised, and any deletion peptides which formed could be eliminated more readily during the subsequent purification procedures. In addition, previous difficulties in purification and characterisation of MAP construct by, e.g. electrospray mass spectroscopy (ES-MS), often led to the multi-dendritic peptidic immunogens being used without full characterisation after dialysis and recovery of the product(s). This article describes an enzymatic (tryptic) digestion method with the MAP construct, followed by characterisation of the enzymatic digest by reversed phase high-performance liquid chromatography-ES-MS. With this method, fragments of the MAP construct cleaved at specific amino acid residue sites (e.g. lysine or arginine) within the sequence of the parent peptide can be readily determined and the kinetics of the digestion easily followed. This enzymatic digestion procedure thus provides a facile approach to confirm that all of the multi-dendritic arms of the purified MAP construct have been equivalently elongated during the peptide synthesis and that consequently the purified construct structure contains the correct peptide sequence.