Fast atom bombardment combined with tandem mass spectrometry for determining structures of small oligonucleotides

A study of small (n = 3 to 6) oligonucleotide and the metastable and collisionally activated decompositions of their (M-H)- species desorbed by using fast atom bombardment (FAB) is reported. Data were obtained for both ribo- and 2'-deoxyribotrinucleotides and for 2'-deoxyribotetra-, penta-, and hexanucleotides. The favored metastable decompositions of all of the oligonucleotides studied are eliminations of neutral CONH and loss of BH, where B is the base moiety. The BH elimination, however, provides little sequence information in the higher oligonucleotides and the process is more indicative of the different bases present in the oligomer. The chemistry observed upon collisional activation changes as one goes from trinucleotides to hexanucleotides. The formation of sequence ions is more facile for processes involving the 3' terminus, allowing the sequence to be determined. As one goes to the higher oligonucleotides, however, several different competitive fragmentation processes become as facile as or more facile than the reactions giving the sequence ions. This hinders proper ion assignments and makes sequence determination difficult.     

Analysis of the distribution of the nucleotide sequence and electrostatic potential of the Escherichia coli genome

The oligonucleotide composition of the E. coli genome and its sigma70-specific promoters has been analyzed. The promoter DNA was shown to contain mainly AT-rich hexanucleotides having functionally important physical properties such as the ability to form easily melting sites and induce the bending of the double helix. A comparative analysis of the electrostatic characteristics of hexanucleotides within the whole sequence of the E. coli genome and its promoter regions was made. Hexanucleotides possessing a more electronegative surrounding were found to predominate in the nucleotide sequence of the promoter DNA.     

Analysis of the nucleotide sequence and electrostatic potential distribution in the <Emphasis Type="Italic">Escherichia coli</Emphasis> genome

Analysis of the oligonucleotide composition of the complete E. coli genome and its σ⁷⁰-specific promoters shows that promoter DNA mainly contains AT-rich hexanucleotides that have functionally important physical properties (propensity to form ‘low-melting’ regions and helix bends). Comparative analysis of the electrostatic characteristics reveals that hexanucleotides corresponding to more electronegative surroundings are mostly found in promoter DNA.     

Synthesis of oligonucleotides with sequences identical with or analogous to the 3''-end of 16S ribosomal RNA of Escherichia coli: preparation of m-6-2-A-C-C-U-C-C and A-C-C-U-C-m-4-2C via phosphotriester intermediates.

The synthesis of two fully-protected hexanucleotides (11a and 11b) via a phosphotriester approach, which is based on the use of two types of protecting groups for the internucleotide linkages, i.e. one 2,2,2-tribromo-ethyl at the 5'-terminus and four 2-chlorophenyl groups for the remaining linkages, is reported. The hexanucleotides 11a and 11b, assembled via a block-wise two-step phosphotriester method, can be deblocked conveniently to give the two hexamers 12a and 12b containing only 3'leads to5' internucleotide linkages.     

A theoretical investigation on the sequence selective binding of daunomycin to double-stranded polynucleotides

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self-complementary double-stranded hexanucleotides: d(CGTACG)2,d(CGATCG)2,d(CITACI)2, d(TATATA)2, d(CGCGCG)2 and d(TACGTA)2. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)2-DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)2 and d(CGCGCG)2, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)2 and d(CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)2 complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent.(ABSTRACT TRUNCATED AT 250 WORDS)     

A theoretical investigation on the sequence selective binding of adriamycin to double-stranded polynucleotides.

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of adriamycin (ADM) to five self-complementary double-stranded hexanucleotides. Among the two regularly alternating hexanucleotides d (TATATA)2 and d (CGCGCG)2, a stronger binding is predicted for the former. The strongest complex is computed, however, for the mixed hexanucleotide d (CGTACG)2, containing the intercalation site between two CG base pairs and an adjacent TA base pair. The overall sequence preference is the result of an intricate interplay of sequence preferences of the constituents in particular of daunosamine and the 9-OH substituent. Altogether, the selective base pair recognition by adriamycin cannot be defined in terms of the two base pairs implicated in the intercalation site alone but must be expressed in terms of a triplet of base pairs.     

Prediction of oligonucleotide frequencies based upon dinucleotide frequencies obtained from the nearest neighbor analysis.

A statistical method of predicting hexanucleotide frequencies is presented. The method requires dinucleotide frequencies which can be readily obtained by nearest neighbor analysis. The frequencies of 64 hexanucleotides of E. coli were estimated and compared well with those predicted by a third order Markov chain.     

A method for the synthesis of oligonucleotide by single addition of 2'-O-(o-nitrobenzyl)nucleoside 5'-diphosphates using polynucleotide phosphorylase.

Two hexanucleotides A-U-G-U-G-A and C-A-A-U-U-G were synthesized from the chemically synthesized trimers C-A-A and A-U-G by addition of 2'-O-(o-nitrobenzyl)nucleoside diphosphates using polynucleotide phosphorylase isolated from either Escherichia coli or Micrococcus luteus. In each reaction the preference of the enzyme was tested. The o-nitrobenzyl group was removed after addition of the mononucleotide and the deblocked product was isolated by chromatography on DEAE-Sephadex in high yields.     

A Theoretical Investigation on the Sequence Selective Binding of Daunomycin to Double-Stranded Polynucleotides

Abstract

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self- complementary double-stranded hexanucleotides: d(CGTACG)₂, d(CGATCG)₂, d(CITACI)₂, d(TATATA)₂, d(CGCGCG)₂ and d(TACGTA)₂. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)₂- DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)₂ and d(CGCGCG)₂, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT)-poly(dA-dT) and poly(dG-dC)<<poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)₂ and d(CGTACG)₂, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)₂ complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent. Altogether, it is seen that the selective base pair recognition by daunomycin cannot, in general, be defined in terms of the two base pairs implicated in the intercalation site alone (with the exception of homogeneous AT or GC base sequences) but must be expressed in terms of a triplet of base pairs.     

Studies of the catalytic properties of an endonuclease isolated from Tetrahymena pyriformis.

An acid endonuclease hydrolyzing both DNA and RNA was purified from Tetrahymena pyriformis, strain E. The enzyme is distributed in all major subcellular compartments and is excreted into the growth medium towards the middle of the logarithmic phase. It hydrolyzes DNA to penta or hexanucleotides, on the average, bearing the monoesterified phosphate at the 3'-position. Particularly in early phases of the reaction it shows a very pronounced specificity for bases with a keto group at position 4 of the pyrimidine ring, such as guanine and thymine.     

Mono- through hexanucleotide composition of the Escherichia coli genome: a Markov chain analysis.

Several statistical methods were tested for accuracy in predicting observed frequencies of di- through hexanucleotides in 74,444 bp of E. coli DNA. A Markov chain was most accurate overall, whereas other methods, including a random model based on mononucleotide frequencies, were very inaccurate. When ranked highest to lowest abundance, the observed frequencies of oligonucleotides up to six bases in length in E. coli DNA were highly asymmetric. All ordered abundance plots had a wide linear range containing the majority of the oligomers which deviated sharply at the high and low ends of the curves. In general, values predicted by a Markov chain closely followed the overall shape of the ordered abundance curves. A simple equation was derived by which the frequency of any nucleotide longer than four bases in the E. coli genome (or any genome) can be relatively accurately estimated from the nested set of component tri- and tetranucleotides by serial application of a 3rd order Markov chain. The equation yielded a mean ratio of 1.03 +/- 0.94 for the observed-to-expected frequencies of the 4,096 hexanucleotides. Hence, the method is a relatively accurate but not perfect predictor of the length in nucleotides between hexanucleotide sites. Higher accuracy can be achieved using a 4th order Markov chain and larger data sets. The high asymmetry in oligonucleotide abundance means that in the E. coli genome of 4.2 X 10(6) bp many relatively short sequences of 7-9 bp are very rare or absent.     

基于Roche 454 GS FLX高通量测序的叶城沙蜥基因组微卫星特征分析

叶城沙蜥Phrynocephalus axillaris是我国特有的一种小型爬行动物,广泛分布于新疆塔里木盆地、吐鲁番-哈密盆地和甘肃敦煌盆地。本研究利用Roche 454 GS FLX高通量测序技术进行叶城沙蜥微卫星位点筛选,获得了91 190条高质量序列。用Krait搜索微卫星位点,共得到1～6个碱基重复类型的完美型微卫星序列29 890个。不同类型微卫星中,单碱基重复类型数目最多,有14 630个,占总数的48. 95%,其次是二碱基,约占28. 60%,四碱基、三碱基、五碱基和六碱基分别占10. 73%、10. 48%、0. 92%和0. 32%。二碱基微卫星中AC重复类型数量最多,三碱基、四碱基、五碱基和六碱基中分别是ATC、AAAT、AAAAT和AATCCC。叶城沙蜥完美型微卫星中数量最多的11种重复拷贝类型分别为C、A、AC、AG、AAAT、ATC、AT、AAT、ATAG、AGG和AAC。本研究深化了对叶城沙蜥基因组的了解,并为以后开发和筛选大量高质量微卫星标记提供了数据支持,也为利用微卫星标记研究叶城沙蜥种群遗传结构和谱系地理模式奠定了基础。     

Structural and thermodynamic analysis of the conformational states of self-complementary hexanucleotides 5′-d(GCATGC) and 5′-d(GCTAGC) in Aqueous Solution

The conformational states of hexanucleotides 5′-d(GCATGC) and 5′-d(GCTAGC) capable of forming hairpins in aqueous solution were studied by 1D and 2D ¹H NMR and molecular dynamics. The equilibrium thermodynamic parameters were determined for the formation of duplexes and hairpins, and the spatial structures were computed for the GCATGC and GCTAGC conformers. The mobility of the hexamer constituents was evaluated by nanosecond molecular dynamics simulation. The possible causes of the observed difference in the thermodynamic stability of the duplex and the hairpin are discussed.     

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.     

Purification and properties of a 3'-phosphoryl former endodeoxyribonuclease from eggs of Asterias forbesi.

A DNA endonuclease has been purified from eggs of Asterias forbesi by a simple four-step-purification procedure. The purified enzyme is at least 96% pure and is free of phosphatase, phosphodiesterase, and RNase. It has a pH optimum of 6.5 and does not require divalent cations. The enzyme produces 3'-phosphoryl and 5'-hydroxyl end groups. The products of exhaustive hydrolysis can be grouped in two fractions. The first fraction, 40%, contains a small amount of mononucleotides and di-, tri-, tetra-, penta-, and hexanucleo-tides. The second fraction, 60%, contains oligonucleotides larger than hexanucleotides.     

Relaxation of recognition sequence of specific endonuclease HindIII.

Under the standard reaction conditions, the restriction endonuclease HindIII cleaves double-stranded DNA, within the recognition sequence--A/AGCTT--at the position indicated by the arrow. In the presence of dimethyl sulfoxide the substrate specificity of this enzyme is reduced and cleavages occur at additional sites. We have determined the secondary sites in pBR322 DNA recognized by HindIII endonuclease under relaxed conditions and found that it cleaves the hexanucleotides: G/AGCTT, A/GGCTT, A/TGCTT, A/ATCTT, A/AGCCT, A/AGCAT, A/AGCGT, A/AGCTC, at the positions indicated by the arrows, producing fragments with cohesive ends.