Sequencing oligonucleotides with blocked termini using exonuclease digestion and electrospray mass spectrometry

A method for sequencing ODNs with both termini blocked using mass spectrometry (MS) is reported. The ladder sequencing method is based on our investigation and understanding of critical factors affecting snake venom phosphodiesterase (SVP) digestion of such ODNs. To produce sequence ladders suitable for MS analysis, digestion conditions such as SVPs from three snake species and pH values of digestion buffer were investigated. SVP of Crotalus duressus terrificus (SVP I) was found to be the most suitable for sequencing ODNs with both termini blocked. The pH value of 9.4, which is optimal for SVP digestion of unmodified ODNs, was found to be unsuitable for ladder sequencing ODNs with both termini blocked. Instead, digestion in a wide range of pH values (pH 5-8), including rarely used acidic conditions, was found to be necessary to obtain otherwise unobtainable sequence information. With digestion buffer of desired pH values, sequence ladders which are recorded as MWs of truncated ODNs from SVP digestion are obtained. Examples of sequencing ODNs up to 26 bases long with both termini blocked are demonstrated in this work.     

Preparation of an imidazole-conjugated oligonucleotide

Postsynthetic modification of an oligonucleotide with an imidazole functional group was achieved by formation of an amide bond between the functional group and a single 2'-amino-2'-deoxyuridine, d-aU, of the oligonucleotide. The succinimidyl ester of N-glutaryl-histamine was synthesized under anhydrous conditions and added to the oligonucleotide in an acetonitrile-containing buffer at pH 8.0. Formation of the conjugate was assayed by digestion with snake venom phosphodiesterase and bacterial alkaline phosphatase, followed by reversed-phase HPLC to resolve constituent nucleosides. The disappearance of a peak corresponding to d-aU and the appearance of a peak that coelutes with authentic 2'-(N-glutaryl-N'-histaminyl)-2'-deoxyuridine confirmed the formation of the conjugate. Imidazole-conjugated oligonucleotides may have utility as antisense agents capable of hydrolyzing RNA.     

Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).     

A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma-optical emission spectroscopy

A novel approach for quantitation of DNA (oligonucleotides) with an unprecedented accuracy of approximately 1% is reported. Quantitation of DNA is commonly performed by measuring UV absorption or fluorescence from dyes intercalated into DNA. Both methods need accurate quantitation standards to yield more comparable results between laboratories. For establishing technically authentic standards for DNA quantitation, a new measurement approach carrying an inherent capability of absolute quantitation is demanded. The proposed approach is based on the stoichiometric existence of phosphorus (P) in DNA. The quantity of P from the phosphodiester backbone of a purified oligonucleotide was accurately determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES) with yttrium internal standard via acid digestion. The number of moles of oligonucleotides was then calculated from that of P using the stoichiometry. The major issues regarding the validity of the suggested approach were (i) effective removal of extra P sources, (ii) quantitative recovery of P through the digestion process, and (iii) oligomeric purity of the target oligonucleotide. These issues were investigated experimentally using various analytical techniques such as matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), capillary electrophoresis, electrical conductometry, UV spectrometry, and gravimetry. In conclusion, it is feasible to certify pure oligonucleotide reference materials with uncertainties less than 1% using the proposed approach.     

Poly(adenosine diphosphate-ribosylation) of nuclear matrix proteins in alkylating agent resistant and sensitive cell lines

Using Walker 256 breast carcinoma cell lines either with or without acquired resistance to alkylating agents, the structural framework proteins of the nucleus, the nuclear matrix proteins, were found to be effective acceptors for poly(ADP-ribose). Incubation of isolated nuclei with nicotinamide adenine [32P] dinucleotide ([32P] NAD), followed by the isolation of the nuclear matrix, demonstrated that two polypeptides of approximate molecular weight (Mr) 105 000 and 116 000 were extensively poly(ADP-ribosylated). By an in vitro [32P] NAD assay, the nuclear matrix fraction was found to maintain approx. 15% of the total nuclear matrix activity of poly(ADP-ribose) polymerase. Confirmation that the trichloroacetic acid (TCA) precipitable material represented ADP-ribose units was achieved by enzymatic digestion of the nuclear matrix preparation with snake venom phosphodiesterase (SVP). Within 15 min, greater than 85% of the 32P label was digested by SVP and the final digestion products were found to be phosphoribosyl-AMP (PR-AMP) and adenosine 5'-monophosphate (5'-AMP) by thin layer chromatographic analysis. The average polymer chain length was estimated to be 6-7 ADP-ribose units. Because poly(ADP-ribose) polymerase has a putative role in DNA repair, a comparison of the nuclear matrix fractions from Walker resistant and sensitive tumor cell lines was made. In both cell lines, the quantitative and qualitative patterns of the nuclear matrix associated poly(ADP-ribosylation) were similar.     

Enhanced activity of an antisense oligonucleotide targeting murine protein kinase C-alpha by the incorporation of 2'-O-propyl modifications.

We have previously described the characterization of a 20mer phosphorothioate oligodeoxynucleotide (ISIS 4189) which inhibits murine protein kinase C-alpha (PKC-alpha) gene expression, both in vitro and in vivo. In an effort to increase the antisense activity of this oligonucleotide, 2'-O-propyl modifications have been incorporated into the 5'- and 3'-ends of the oligonucleotide, with the eight central bases left as phosphorothioate oligodeoxynucleotides. Hybridization analysis demonstrated that these modifications increased affinity by approximately 8 and 6 degrees C per oligonucleotide for the phosphodiester (ISIS 7815) and phosphorothioate (ISIS 7817) respectively when hybridized to an RNA complement. In addition, 2'-O-propyl incorporation greatly enhanced the nuclease resistance of the oligonucleotides to snake venom phosphodiesterase or intracellular nucleases in vivo. The increase in affinity and nuclease stability of ISIS 7817 resulted in a 5-fold increase in the ability of the oligonucleotide to inhibit PKC-alpha gene expression in murine C127 cells, as compared with the parent phosphorothioate oligodeoxynucleotide. Thus an RNase H-dependent phosphorothioate oligodeoxynucleotide can be modified as a 2'-O-propyl 'chimeric' oligonucleotide to provide a significant increase in antisense activity in cell culture.     

Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry

The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.     

Macromolecular derivatives of NAD+ in heart nuclei: poly adenosine diphosphoribose and adenosine diphosphoribose proteins.

Rates of poly (ADP-R) formation from NAD⁺ were determined in isolated pigeon heart and liver nuclei. In heart nuclei K_m for NAD⁺ was 330 μM. On a DNA basis rates were more than twice in heart nuclei than in liver nuclei. The polymer poly (ADP-R) was identified in both nuclear systems by isolation, digestion with snake venom phosphodiesterase and chromatographic separation of phosphoribosyl-AMP and AMP. ADP-R binds to macromolecular nuclear components to form ADP-R derivatives, which upon digestion with snake venom phosphodiesterase yield only AMP, distinguishing these ADP-R compounds from poly (ADP-R).     

Direct determination of DNA nucleotide sequences: structure of a fragment of bacteriophage phiX172 DNA

Methods for the direct determination of nucleotide sequences in DNA have been developed and used to determine the complete primary structure of a fragment of bacteriophage φX174 DNA which is 48 residues in length. This fragment was liberated from φX DNA by digestion at low temperature and high ionic strength with the T4 phage-induced endonuclease IV. The fragment was redigested with endonuclease IV under vigorous conditions and the products fractionated two-dimensionally providing a characteristic endonuclease IV “fingerprint” of the fragment. The Burton (Burton &; Petersen, 1960) depurination reaction was used to characterize the redigestion products and identify the pyrimidine residues at their 5′ and 3′ termini. These oligonucleotide products were then fully sequenced by partial exonuclease digestion with spleen and snake venom phosphodiesterase and analysis of the fractionated digests by base composition, depurination, and 5′ end-group analysis using exonuclease I. Rules for the interpretation of two-dimensional fingerprints of partial exonuclease digests, which rapidly provide sequence information by simple inspection, were also deduced. To derive the complete structure of the fragment, the fully sequenced oligonucleotides were ordered by characterizing large, overlapping, partial endonuclease IV digestion products by means of the depurination reaction. The sequencing methods described are general and may be used for the direct determination of the primary structures of other fragments of DNA.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

A two-dimensional thin layer chromatographic procedure for the sequential analysis of oligonucleotides employing tritium post-labeling.

Two dimensional PEI-cellulose thin layer chromatography can resolve sequentially degraded oligonucleotide fragments of tRNA. This technique entails the sequential degradation of the oligonucleotide with snake venom phosphodiesterase in the presence of bacterial alkaline phosphatase, and periodate oxidation followed by tritiated sodium borohydride reduction of the 3' terminal nucleoside. Subsequently the tritiated oligonucleotide fragments were resolved by two dimensional PEI-cellulose TLC. The results of these experiments indicate that, in some cases, the complete nucleotide sequence of a large oligonucleotide fragment may be determined by interpretation of the observed mobility shifts, thereby eliminiating the need for additional analysis of the oligonucleotide. In addition, the use of two-dimensional rather than one-dimensional resolution of the tritium labeled fragments allows for a complete separation of any interfering background spots from the sequentially degraded oligonucleotides. This procedure was applied to the complete nucleotide sequence analysis of several ribonuclease T1Val and ribonuclease A digestion products from human placenta tRNA.     

A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site.

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability. Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine). This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI. Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III. The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability. Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome. This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.     

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.     

Sequence determination of 5'-terminal and 3'-terminal T1 oligonucleotides of 18-S ribosomal RNA of a mouse cell line (L 5178 Y).

The 5' and 3'-terminal oligonucleotides of 18-S ribosomal RNA of L 5178 Y (a mouse cell line) obtained after total T1 ribonuclease hydrolysis were isolated by a diagonal procedure. They were localized on the fingerprint of T1-ribonuclease-hydrolysed 18-S RNA. These two oligonucleotides were partially hydrolysed by snake venom and spleen phsophodiesterases and resulting products were fractionated bidimensionally. Their base compositions were determined by total hydrolysis with piperidine or snake venom phosphodiesterase. From these results the following sequences were deduced: pU-A-C-C-U-G for the 5'-terminal oligonucleotide and G-A-U-C-A-U-U-Aoh for the 3'-terminal oligonucleotide. Quantitative studies indicated that these sequences represent at least 70% for the 5' oligonucleotide and 85% for the 3' oligonucleotide of the terminal sequences of the 18-S RNA.     

Metabolic stability of 2'' 5''oligo (A) and activity of 2'' 5''oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells.

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.     

Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工(英文)

为研究蛇毒C型凝集素类蛋白的快速进化机制和结构功能关系 ,使用PCR技术扩增了若干编码C型凝集素类蛋白 β链的cDNA分子以及agkisasinβ的基因组DNA ,并将这些扩增产物进行克隆和测序 .对测序结果与试验过程中的具体条件进行了因果关系分析 ,并且进行点阵图比较和多序列比对 .结果表明 ,可能存在“转录后同源重组”等转录后的事件 ,在蛇毒C型凝集素类蛋白的多样性上起着重要的作用 .对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题 ,具有一定的参考价值 .首次报告蛇毒C型凝集素类蛋白的基因组DNA序列 ,其中未发现有内含子     

Use of potato tuber nucleotide pyrophosphatase to synthesize adenosine 5'-monophosphate methyl ester: evidence that the solvolytic preferences of the enzyme are regulated by pH and temperature

Nucleotide alkyl esters are pharmacologically important as potential (ant)agonists of purinoceptors and inhibitors of enzymes. Potato nucleotide pyrophosphatase (PNP) was compared with snake venom phosphodiesterase (SVP) as a catalyst to synthesize nucleotide alkyl esters. In methanol-water mixtures, the methanolysis/hydrolysis ratio of PNP, but not SVP, changed with pH and temperature, being optimal at high pH and low temperature. In a semi-preparative experiment, a crude PNP preparation produced 0.17 mM AMP-O-methyl ester (AMP-OMe) from 1 mM diadenosine 5',5"'-P1,P2-diphosphate (AppA) and 5M methanol, at pH 9 and 0 degrees C. Drawbacks to large-scale use are: low rates inherent to low temperatures, ATP unsuitability as a substrate for alcoholysis, and high cost of AppA. Advantages of PNP vs. SVP are cheapness, non-toxicity, and availability of the enzyme source.