Fluoride-cleavable biotinylation phosphoramidite for 5'-end-labeling and affinity purification of synthetic oligonucleotides

A fluoride-cleavable phosphoramidite for biotinylation was designed, synthesized and coupled efficiently to the 5′-end of DNA on an automatic synthesizer. The diisopropylsilyl acetal functionality was used to link the biotin moiety through a tertiary hydroxide group to the 5′-end of DNA. This linkage proved to be completely stable under certain post-synthetic DNA cleavage/deprotection conditions [0.05 M K₂CO₃ in MeOH, room temperature, 24 h and MeNH₂ (~40%)/NH₄OH (~29%), 1:1 v/v, 65°C, 30 min] while it can be readily broken by fluoride ion, releasing unmodified DNA. To demonstrate the use of this DNA biotinylation method, we applied this method in affinity purification of synthetic DNA. As revealed by HPLC analysis, biotinylated full-length DNA can be efficiently attached to NeutrAvidin™ coated microspheres, and failure sequences can be readily removed. Subsequent treatment of the microspheres with pyridine/HF released high quality full-length unmodified DNA in good yield.     

Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5'' terminus.

A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter.     

Mechanism of ATP-dependent translocation of E.coli UvrD monomers along single-stranded DNA

Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA.     

A simple method for 3'-labeling of RNA.

We describe a simple method for 3'-end labeling RNAs of known sequence. A short DNA template is designed to anneal to the 3'-end of the RNA, with a two nucleotide 5' overhang of 3'-TA-5', 3'-TG-5' or 3'-TC-5'. The Klenow fragment of DNA polymerase I can then cleanly and efficiently extend the 3'-end of the RNA by the incorporation of a single alpha-32P-labeled dATP residue. This method can be used to label one RNA in a mixture of RNAs, or to label 5'-blocked RNAs such as mRNA.     

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.

We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.     

Arthrobacter luteus restriction endonuclease recognition sequence and its cleavage map of SV40 DNA.

The nucleotide sequence at the cleavage site of the restriction endonuclease isolated from Arthrobacter luteus (Alu) has been determined. The endonuclease cleaves at the center of a palindromic tetranucleotide sequence to give even-ended duplex DNA fragments phosphorylated at the 5'-end. The endonuclease cleaves SV40 form I DNA into 32 fragments. The order and sizes of these fragments have been determined to provide an Alu cleavage map of the SV40 genome.     

Sequence-specific labeling of superhelical DNA by triple helix formation and psoralen crosslinking.

Site-specific labeling of covalently closed circular DNA was achieved by using triple helix-forming oligonucleotides 10, 11 and 27 nt in length. The sequences consisted exclusively of pyrimidines (C and T) with a reactive psoralen at the 5'-end and a biotin at the 3'-end. The probes were directed to different target sites on the plasmids pUC18 (2686 bp), pUC18/4A (2799 bp) and pUC1 8/4A-H 1 (2530 bp). After triple helix formation at acid pH the oligonucleotides were photocrosslinked to the target DNAs via the psoralen moiety, endowing the covalent adduct with unconditional stability, e.g. under conditions unfavorable for preservation of the triplex, such as neutral pH. Complex formation was monitored after polyacrylamide gel electrophoresis by streptavidin-alkaline phosphatase (SAP)-induced chemiluminescence. The yield of triple helix increased with the molar ratio of oligonucleotide to target and the length of the probe sequence (27mer > 11mer). The covalent adduct DNA were visualized by scanning force microscopy (SFM) using avidin or streptavidin as protein tags for the biotin group on the oligonucleotide probes. We discuss the versatility of triple helix DNA complexes for studying the conformation of superhelical DNA.     

Determination of kanamycin in serum by solid-phase extraction,pre-capillary derivatization and capillary electrophoresis

An effective method based on solid-phase extraction (SPE) and capillary electrophoresis (CE) for the determination of kanamycin in human serum was developed and validated. Off-line SPE was employed for the isolation of kanamycin from serum on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge. A mixture of 0.2 M borate (pH 10.5)-methanol (50:50, v/v) was used as analyte eluting solvent. After pre-capillary derivatization with o-phthalaldehyde/mercaptoacetic acid reagent, the sample was analyzed by CE with a separation buffer of 30 mM borax, pH 10.0, containing 16% (v/v) methanol. A linear response over the concentration range 5-40 microgram/ml was obtained with a detection limit of 2 microgram/ml. Intra-day and inter-day precision were 6.2 and 10.3% RSD, respectively. Recoveries of approximately 90% were found. For the determination of lower levels of kanamycin (<5 microgram/ml), NH(4)OH (25%, w/v)-methanol (30:70, v/v) was used for analyte elution. After evaporation, reconstitution and derivatization, the sample was analyzed by on-line field-amplified sample stacking (FASS) CE. Good linearity in the concentration range 0.4-5 microgram/ml was obtained with a detection limit of 0.1 microgram/ml. Intra-day and inter-day RSD were 3.4 and 11.2%, respectively. Recoveries of approximately 60% were found. The method was successfully applied to the analysis of kanamycin in sera of tuberculosis patients at peak level and trough level concentrations.     

Separation and determination of astaxanthin from microalgal and yeast samples by molecularly imprinted microspheres

In this work, molecularly imprinted microspheres (MIMs) were synthesized by aqueous microsuspension polymerization using astaxanthin (3,3'-dihydroxy-beta,beta'-carotene-4,4'-dione) as imprinting molecule. The MIMs obtained were subsequently packed into the stainless steel column and the chromatographic characterization of the column was investigated. The effects of pH and composition of the mobile phase on the retention factor (k') were investigated in detail. The mixture of methanol and dichloromethane (DCM) (8:2, v/v) was used as mobile phase A while the mixture of methanol and water (5:5, v/v) as mobile phase B. The separation of astaxanthin and zeaxanthin (3,3'-dihydroxyl-beta-carotene) was obtained when the concentration of mobile phase B was higher than 30% (v/v) due to their strong lipophilicity. The method developed was successfully applied to separate astaxanthin in the saponified samples of the microalga Haematococcus pluvialis and the yeast Phaffia rhodozyma. The recovery of adding 40 mg astaxanthin to 1.0 g microalgal sample was 95.5% with an R.S.D. (n =5) of 5.3%. The results of determination of astaxanthin in the microalga and the yeast were 3.7% (R.S.D (n = 1.5%, n = 9) and 0.041% (R.S.D n= 7.3%, n = 9), respectively.     

Quantification of polymerase chain reaction products by affinity-based hybrid collection.

We have used oligonucleotides modified with biotin in the 5'-end as primers in the polymerase chain reaction (PCR)-amplification. This results in the synthesis of 5'-biotinylated DNA molecules, which are detected by hybridization to a labelled probe in solution. The formed hybrids are collected on an avidin-matrix by mediation of the biotin residue of the target molecules. The affinity-based hybrid collection method is quantitative and makes it possible to measure the amount of DNA produced in the PCR-amplification. At low concentrations of template the efficiency of the process is close to 100%, making it possible to detect the presence of a few molecules of target DNA in 25 cycles. With high template concentrations the efficiency of the process is low.     

生物可降解微球作为乙型肝炎基因免疫佐剂的研究

探讨生物可降解微球对基因免疫的增强作用。采用有机溶剂蒸发法制备聚乳酸聚乙醇酸共聚 物（ＰＬＧＡ）微球,构建含有乙型肝炎病毒表面抗原Ｓ基因的ｐＲＣ－ＣＭＶ真核表达载体,用微球与基因 载体共孵育法制备其混合物。肌肉注射免疫Ｂａｌｂ／ｃ小鼠。结果表明：微球注射组的血清抗体滴度达到 ｌ：１６００,其效果与乙型肝炎病毒表面抗原加铝佐剂注射组相近,而裸ＤＮＡ注射组没有反应。说明了 生物可降解微球可显著的提高基因免疫的免疫反应。     

Hepatitis C virus NS3 and simian virus 40 T antigen helicases displace streptavidin from 5'-biotinylated oligonucleotides but not from 3'-biotinylated oligonucleotides: evidence for directional bias in translocation on single-stranded DNA

Helicases are enzymes that use energy from nucleoside triphosphate hydrolysis to unwind double-stranded (ds) DNA, a process vital to virtually every phase of DNA metabolism. Helicases have been classified as either 5'-to-3' or 3'-to-5' on the basis of their ability to unwind duplex DNA adjacent to either a 5' or 3' single-stranded (ss) DNA overhang. However, there has been debate as to whether this substrate preference is indicative of unidirectional translocation on ssDNA. We developed an assay that monitors the ability of a helicase to displace streptavidin from biotinylated oligonucleotides [Morris, P. D., and Raney, K. D. (1999) Biochemistry 38, 5164-5171]. Two helicases identified as having 5'-to-3' polarity displaced streptavidin from the 3'-end of biotinylated oligonucleotides but not from the 5'-end. We performed similar experiments using the 3'-to-5' helicases from the hepatitis C virus (NS3) and SV40 virus (SV40 T antigen). NS3 and SV40 T antigen were able to displace streptavidin from a 5'-biotinylated oligonucleotide but not from a 3'-biotinylated oligonucleotide. NS3 and SV40 T antigen enhanced the spontaneous rate of dissociation of streptavidin from biotin 340-fold and 1700-fold, respectively. The ssDNA binding protein, gp32, did not enhance dissociation of streptavidin from either end of an oligonucleotide. For NS3, the rate of displacement was faster from a 5'-biotinylated 60mer than from a 5'-biotinylated 30mer. The strong directional bias in streptavidin displacement activity exhibited by each helicase is consistent with a directional bias in translocation on ssDNA. The dependence of the reaction with NS3 on the oligonucleotide length suggests that multiple NS3 monomers are necessary for optimal activity.     

Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.

The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.     

The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5- to 10-fold over the control and is extremely potent, exerting a saturating response at a a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids in 5-fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.     

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5'-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5'-end of the gene and the 3'-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.     

Adenovirus DNA synthesized in the presence of aphidicolin

Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals.