Ligase joining of oligodeoxynucleotides on a oligodeoxynucleotide analog matrix containing P-S-C(5')-internucleotide bonds

Joining of oligodeoxynucleotides by T4 polynucleotide ligase on an oligodeoxynucleotide template analogue having P--S--C(5') internucleotide bonds was investigated. The enzyme did ligate the oligodeoxynucleotides but with an extremely low rate. The major product of the enzymatic reaction proved to be the adenylylated intermediate of the 5'-phosphorylated oligodeoxynucleotide, the product of the second stage of the overall ligation reaction. It is noteworthy that this intermediate is not accumulated in ligase reactions under standard conditions when DNA templates rather than template analogues were used.     

Synthesis of oligodeoxyribonucleotides containing 5-fluoro-2'-deoxyribocytidine]

A synthesis of the phosphoroamidite derivative of 5-fluoro-2'-deoxycytidine which allows one to introduce the modified nucleoside residue into the 5'-position of the oligodeoxyribonucleotide by the standard solid phase phosphoroamidite method, has been carried out. Oligonucleotides with 5-fluoro-2'-deoxycytidine residues in various positions of the DNA strand are obtained by the combination of chemical and enzymatic syntheses.     

A facile synthesis of 5'-end solid-anchored, 3'-end free oligodeoxyribonucleotides via the (5'-->3')-elongated phosphoramidite strategy

It is demonstrated that not only N2- but also O6-protection of the guanine base is necessary for obtaining the oligodeoxyribonucleotides in high yields and at a high purity in the solid-phase synthesis via the (5'--> 3')-chain elongated phosphoramidite approach.     

Synthesis and antisense properties of oligodeoxyribonucleotides containing C5-substituted arabinofuranosyluracil

An oligodeoxyribonucleotide (ODN) containing three C5-substituted arabinofuranosyluracils was synthesized by the post-synthetic modification method from the ODN containing three C5-substituted 2,2'-anhydrouridines. The stability of the modified ODN/DNA duplex was lower than that of the corresponding normal duplex but that of the modified ODN/RNA duplex showed little change. The modified ODN could induce RNase H activity and was resistant against nuclease.     

Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

The structure of d(TpA), the major photoproduct of thymidylyl-(3'5')-deoxyadenosine.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.     

Lsm proteins bind and stabilize RNAs containing 5' poly(A) tracts

Many orthopoxvirus messenger RNAs have an unusual nontemplated poly(A) tract of 5 to 40 residues at the 5' end. The precise function of this feature is unknown. Here we show that 5' poly(A) tracts are able to repress RNA decay by inhibiting 3'-to-5' exonucleases as well as decapping of RNA substrates. UV cross-linking analysis demonstrated that the Lsm complex associates with the 5' poly(A) tract. Furthermore, recombinant Lsm1-7 complex specifically binds 5' poly(A) tracts 10 to 21 nucleotides in length, consistent with the length of 5' poly(A) required for stabilization. Knockdown of Lsm1 abrogates RNA stabilization by the 5' poly(A) tract. We propose that the Lsm complex simultaneously binds the 3' and 5' ends of these unusual messenger RNAs and thereby prevents 3'-to-5' decay. The implications of this phenomenon for cellular mRNA decay are discussed.     

The structure and application of oligodeoxyribonucleotides containing modified, degenerate bases.

Oligodeoxynucleotides containing modified pyrimidine bases which can stably hydrogen-bond to adenine and guanine and also purine bases which pair with thymine and cytosine residues in duplexes have been synthesised, as have oligomers with both such analogues. Structures have been investigated by melting transitions, n.m.r. spectroscopy and crystallography and then interpreted in terms of tautomeric equilibria. Applications to hybridisation probes and primers will be discussed.     

Synthesis and structure of new phosphines containing P-N bonds

New phosphines containing the homopiperazine backbone have been prepared. The X-ray structure of the P(III) phosphine and its P(V) sulphide are compared. The P-N bond length is shortened on oxidation from P(III) to P(V).     

5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses.

Within the conserved 5' untranslated region (UTR) of the pestivirus genome three highly variable regions were identified. Preceding the polyprotein start codon, multiple cryptic AUG codons and several small open reading frames are characteristic for all the five pestiviruses. Inspection of the context of AUGs revealed that the polyprotein initiation AUG of pestivirus has a weak context for efficient translation initiation. The most favorable context was found in two of the cryptic AUGs. Two oligopyrimidine-rich tracts upstream to the conserved either cryptic or authentic AUG in the 5'-UTR of pestivirus were identified and 83.3% of their nucleotide sequences are complementary to the consensus sequence at the 3' terminus of eucaryotic 18S rRNA. A secondary structure model for the 5'-UTR of pestivirus was predicted. Nucleotide sequence comparison among five pestiviruses led to the identification of a variable region and a conserved region in the 3'-UTR. A deletion of 41 nucleotides was found within the variable region in Osloss. A secondary structure model for the 3'-UTR was also predicted. The structural similarity of the 5'-UTR between pestiviruses and picornaviruses and hepatitis C viruses was demonstrated and the possible implications of features of the 5' and 3'-UTR of pestiviruses are discussed.     

Synthesis of 5'(3')-O-amino nucleosides

Synthetic methods leading to 5'(3')-O-amino nucleosides have been developed in an effort to prepare derivatives that may have antitumor or antiviral activities. They are based on ring opening of O2,5'-cyclonucleosides with the N-protected hydroxylamines and dehydrative coupling of 5'(3')-O-unprotected nucleosides with N-hydroxyphthalimide.     

Rapid amplification of 5' complementary DNA ends (5' RACE)

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.     

The secondary structure of nucleotidyl-(5' bound to N)-amino acids containing different heterocyclic bases and amino acids]

Nucleotidyl-(5' leads to N)-amino acids containing different heterocycle bases: adenine, guanine, hypoxanthine, cytosine, uracyl, and aromatic amino acids: phenylalanine, tyrosine and tryptophan, have been investigated by proton magnetic resonance and circular dichroism. For all the compounds studied folded conformation have been shown stabilized by hydrophobic interaction in aqueous solution. The comparison of the results of the studied nucleotidyl-(5' leads to N)-amino acids unable us to build four secondary structure types in these very compounds. Phenylalanine and tyrosine derivatives of purine nucleotides can be regarded as the first type, tryptophan derivatives of purine nucleotides as the second type, phenylalanine and tyrosine derivatives of pyrimidine nucleotides as the third type and tryptophan derivatives of pyrimidine nucleotides as the fourth type. For each group of these compounds conformational models have been built. In all these compounds the anti-conformation has been proved to exist.     

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor,target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.     

Introduction of tri- and tetraphosphate internucleotide bonds into oligodeoxyribonucleotides

Dodecadeoxyribonucleotides d(AGCTTGpppGCTGCA) and d(AGCTTGppppGCTGCA) were obtained by template directed chemical condensation induced by a water-soluble carbodiimide.     

The structure of cytidilyl(2',5')adenosine when bound to pancreatic ribonuclease S

The three-dimensional structure of the RNase S complex with the synthetic dinucleoside monophosphate cytidilyl(2′,5′)adenosine(C₂,p₅,A) is determined using difference Fourier techniques at 2.0 Å resolution in conjunction with computer graphic model-building and energy minimization. The latter has been carried out as a function of the rigid body parameters of the dinucleoside monophosphate and the dihedral angles of the nucleoside portion as well as of relevent amino acids in the active site of the enzyme.The bound dinucleoside monophosphate is found to assume an extended conformation, with the adenine and cytidine bases nearly perpendicular. The bases form specific hydrogen bonds with groups in the active site. Although the atoms involved in the recognition of the pyrimidine base by the enzyme are the same as in the pyrimidine bases of UMP, CMP and UpcA, the details of the binding are different. The adenosine moiety blocks most of the various positions that His119 occupies in the native enzyme and forces it into one well-defined position. One of the His119 ring protons is in contact with O(5′) (the leaving group), O(1′) of the adenine ribose and with a free phosphoryl oxygen. No strong charge contacts with the phosphate group are observed.We show how combining X-ray data with computer graphic model-building, electron density fitting and energy calculations leads to the model we propose and discuss in detail the enzyme-nucleic acid interactions.