Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine

The replacement of thymidine with 5-bromo-2′-deoxyuridine (^BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a ^BrdU or its closely related 5-bromo-2′-deoxycytidine (^BrdC). Our results showed that two types of crosslink products could be induced from d(^BrCG), d(^BrUG), d(G^BrU), or d(A^BrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5′ flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5′ neighboring nucleoside was a dG than while it was a dA, and ^BrdC induced the formation of crosslink products much more efficiently than ^BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.     

Structural basis for uracil DNA glycosylase interaction with uracil: NMR study

Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5′ end of the tetraloop. The ¹H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T₁₂ and T₁₅ nucleotides stack upon 3′ and 5′ ends of the stem, respectively. Further, T₁₄ stacks upon both T₁₂ and T₁₅. Though U₁₃ partially stacks upon T₁₄, no stacking interaction is observed between U₁₃ and T₁₂. All the individual nucleotide bases belonging to the stem and T₁₂ and T₁₅ of the loop adopt ‘anti’ conformation with respect to their sugar moiety, while the U₁₃ and T₁₄ of the loop are in ‘syn’ conformation. The turning phosphate in the loop is located between T₁₃ and T₁₄. This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5′ end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The of T₁₂ and α, β, γ, and ζ of U₁₃ and γ of T₁₄, which partially influence the local conformation of U₁₃ in U2-hairpin are all locked in ‘trans’ conformation. Such stretched out backbone conformation in the vicinity of U₁₃ could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5′ end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.     

UVA irradiation of BrU-substituted DNA in the presence of Hoechst 33258

Given that our knowledge of DNA repair is limited because of the complexity of the DNA system, a technique called UVA micro-irradiation has been developed that can be used to visualize the recruitment of DNA repair proteins at double-strand break (DSB) sites. Interestingly, Hoechst 33258 was used under micro-irradiation to sensitize 5-bromouracil (^BrU)-labelled DNA, causing efficient DSBs. However, the molecular basis of DSB formation under UVA micro-irradiation remains unknown. Herein, we investigated the mechanism of DSB formation under UVA micro-irradiation conditions. Our results suggest that the generation of a uracil-5-yl radical through electron transfer from Hoechst 33258 to ^BrU caused DNA cleavage preferentially at self-complementary 5′-AA^BrU^BrU-3′ sequences to induce DSB. We also investigated the DNA cleavage in the context of the nucleosome to gain a better understanding of UVA micro-irradiation in a cell-like model. We found that DNA cleavage occurred in both core and linker DNA regions although its efficiency reduced in core DNA.     

Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (^NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (^NU_m), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of ^NU and ^NU_m substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either ^NU or ^NU_m and of DNA:RNA hybrid duplexes containing ^NU_m. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to ^NU, ^NU_m decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the ^NU_m containing DNA:DNA duplex structure. The conformation of ^NU_m, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of ^NU_m to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.     

Photoreactivities of 5-Bromouracil-containing RNAs

5-Bromouracil (^BrU) was incorporated into three types of synthetic RNA and the products of the photoirradiated ^BrU-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA^BrUGC)₂ and r(CGAA^BrUUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine (^ketoA) product at the 5′-neighboring nucleotide, such as r(GC^ketoAUGC) and r(CGA^ketoAUUGC), respectively. The photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in Z-form RNA produced the 2′-keto guanosine (^ketoG) product r(CGC^ketoGUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.     

Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO₃^·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO₃^·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpC_npT) and 5′-d(TpC_npG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO₃^·− radicals and enzymatic excision of the 5′-d(G^*pT^*) product.     

Recognition of 5-aminouracil (U(#)) in the central strand of a DNA triplex: orientation selective binding of different third strand bases

A necessary feature of the natural base triads for triplex formation is the requirement of a purine (A or G) in the central position, since only these provide sets of two hydrogen bond donors/acceptors in the major groove of the double helix. Pyrimidine bases devoid of this feature have incompatible complementarity and lead to triplexes with lower stability. This paper demonstrates that 5-aminouracil (U^#) (I), a pyrimidine nucleobase analogue of T in which 5-methyl is replaced by 5-amino group, with hydrogen bonding sites on both sides, is compatible in the central position of triplex triad X*U^#·A, where X = A/G/C/T/2-aminopurine (AP), and * and · represent Hoogsteen and Watson–Crick hydrogen bonding patterns respectively. A novel recognition selectivity based on the orientation (parallel/antiparallel) of the third strand purines A, G or AP with A in the parallel motif (A_p*U^#·A), and G/AP in the antiparallel motif (G_ap/AP_ap*U^#·A) is observed. Similarly for pyrimidines in the third strand, C is accepted only in a parallel mode (C_p*U^#·A). Significantly, T is recognised in both parallel and antiparallel modes (T_p/T_ap*U^#·A), with the antiparallel mode being stable compared to the parallel one. The ‘U^#’ triplexes are also more stable than the corresponding control ‘T’ triplexes. The results expand the lexicon of triplex triads with a recognition motif consisting of pyrimidine in the central strand.     

Specificity and Catalytic Mechanism in Family 5 Uracil DNA Glycosylase

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O₂ of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb relies on multiple catalytic residues to facilitate its excision of hypoxanthine and xanthine. This study underscores the structural and functional diversity in the UDG superfamily.     

Rabbit liver tRNAVal1: II. Unusual secondary structure of T ψ C stem and loop due to a U54:A60 base pair

In contrast to all other known tRNAs, mammalian tRNA^Val₁ contains two adenosines A₅₉ and A₆₀, opposite to U₅₄ and ψ₅₅ in the UψCG sequence of the TψC loop, which could form unusual A:U (or A:ψ) pairs in addition to the five “normal” G:C pairs. In order to measure the number of G:C and A:U (A:ψ) pairs in the TψC stem, we prepared the 30 nucleotide long 3′-terminal fragment of this tRNA by “m⁷G-cleavage”. From differentiated melting curves and temperature jump experiments it was concluded that the TψC stem in this fragment is in fact extended by an additional A₆₀:U₅₄ pair. A dimer of this fragment with 14 base pairs was characterized by gel electrophoresis and by the same physical methods. An additional A:U pair in the tRNA^Val₁ fragment does not necessarily mean that this is also true for intact tRNA. However, we showed that U₅₄ is far less available for enzymatic methylation in mammalian tRNA^Val₁ compared to tRNA from T⁻E. coli. This clear difference in U₅₄ reactivity, together with the identification of an extra A₆₀:U₅₄ pair in the UψCG containing fragment suggests the presence of a 6 base pair TψC stem and a 5 nucleotide TψC loop in this tRNA.     

The Putative Nucleic Acid Helicase Sen1p Is Required for Formation and Stability of Termini and for Maximal Rates of Synthesis and Levels of Accumulation of Small Nucleolar RNAs in Saccharomyces cerevisiae

Sen1p from Saccharomyces cerevisiae is a nucleic acid helicase related to DEAD box RNA helicases and type I DNA helicases. The temperature-sensitive sen1-1 mutation located in the helicase motif alters the accumulation of pre-tRNAs, pre-rRNAs, and some small nuclear RNAs. In this report, we show that cells carrying sen1-1 exhibit altered accumulation of several small nucleolar RNAs (snoRNAs) immediately upon temperature shift. Using Northern blotting, RNase H cleavage, primer extension, and base compositional analysis, we detected three forms of the snoRNA snR13 in wild-type cells: an abundant TMG-capped 124-nucleotide (nt) mature form (snR13F) and two less abundant RNAs, including a heterogeneous population of ~1,400-nt 3′-extended forms (snR13R) and a 108-nt 5′-truncated form (snR13T) that is missing 16 nt at the 5′ end. A subpopulation of snR13R contains the same 5′ truncation. Newly synthesized snR13R RNA accumulates with time at the expense of snR13F following temperature shift of sen1-1 cells, suggesting a possible precursor-product relationship. snR13R and snR13T both increase in abundance at the restrictive temperature, indicating that Sen1p stabilizes the 5′ end and promotes maturation of the 3′ end. snR13F contains canonical C and D boxes common to many snoRNAs. The 5′ end of snR13T and the 3′ end of snR13F reside within C₂U₄ sequences that immediately flank the C and D boxes. A mutation in the 5′ C₂U₄ repeat causes underaccumulation of snR13F, whereas mutations in the 3′ C₂U₄ repeat cause the accumulation of two novel RNAs that migrate in the 500-nt range. At the restrictive temperature, double mutants carrying sen1-1 and mutations in the 3′ C₂U₄ repeat show reduced accumulation of the novel RNAs and increased accumulation of snR13R RNA, indicating that Sen1p and the 3′ C₂U₄ sequence act in a common pathway to facilitate 3′ end formation. Based on these findings, we propose that Sen1p and the C₂U₄ repeats that flank the C and D boxes promote maturation of the 3′ terminus and stability of the 5′ terminus and are required for maximal rates of synthesis and levels of accumulation of mature snR13F.     

C5'- and C3'-sugar radicals produced via photo-excitation of one-electron oxidized adenine in 2'-deoxyadenosine and its derivatives

We report that photo-excitation of one-electron-oxidized adenine [A(-H)•] in dAdo and its 2′-deoxyribonucleotides leads to formation of deoxyribose sugar radicals in remarkably high yields. Illumination of A(-H)• in dAdo, 3′-dAMP and 5′-dAMP in aqueous glasses at 143 K leads to 80-100% conversion to sugar radicals at C5′ and C3′. The position of the phosphate in 5′- and 3′-dAMP is observed to deactivate radical formation at the site of substitution. In addition, the pH has a crucial influence on the site of sugar radical formation; e.g. at pH ~5, photo-excitation of A(-H)• in dAdo at 143 K produces mainly C5′• whereas only C3′• is observed at high pH ~12. ¹³C substitution at C5′ in dAdo yields ¹³C anisotropic couplings of (28, 28, 84) G whose isotropic component 46.7 G identifies formation of the near planar C5′•. A β-¹³C 16 G isotropic coupling from C3′• is also found. These results are found to be in accord with theoretically calculated ¹³C couplings at C5′ [DFT, B3LYP, 6-31(G) level] for C5′• and C3′•. Calculations using time-dependent density functional theory [TD-DFT B3LYP, 6-31G(d)] confirm that transitions in the near UV and visible induce hole transfer from the base radical to the sugar group leading to sugar radical formation.     

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA–protein complexes at several 5′ splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5′ splice site (5′ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5′ss of TSHβ exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.     

Poly(U) RNA-templated synthesis of AppA

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5′-5′ AppA synthesis by 3′-5′ poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K⁺) and (Mg²⁺) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pK_a’s. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5′-5′ joining between stacked or concurrently paired A''s can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5′-5′ purine dinucleotides might be readily synthesized from 3′-5′ RNAs with complementary sequences.     

Novel Pt(II) and Pt(IV) complexes with 3-amino-5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione. Synthesis, physicochemical, chemometric and pharmacological investigation

Platinum(II) and platinum(IV) complexes with 3-amino-5-methyl-5-(4-pyridyl)-2,4-imidazolidenedione (L) with general formulaе cis-[PtL₂X₂]·nH₂O and [PtL₂Cl₄], where X = Cl⁻, Br⁻, I⁻ and n = 2-4) were synthesized. The novel compounds were fully characterized by elemental analysis, IR, ¹H, ¹³C, ¹⁹⁵Pt NMR spectra, thermal analysis and molar conductivity. The geometry of Pt(II) complexes and of the organic ligand in the gas phase were optimized using the hybrid DFT method B3LYP with LANL2DZ and 6-31G** basis sets. Some physicochemical parameters as dipole moment, HOMO, LUMO energies and ESP charges were calculated. The comparison of the bond length and angles, obtained from the X-ray analysis and DFT calculations is realized. The cytotoxic effects of these complexes in human T-cell leukemia KE-37 (SKW-3) are reported.     

Synthesis of carbon-11-labeled CK1 inhibitors as new potential PET radiotracers for imaging of Alzheimer’s disease

The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-methoxybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield, respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [¹¹C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([¹¹C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[¹¹C]methoxybenzamide ([¹¹C]5c), were prepared from their O-desmethylated precursor 6a or 6b with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–45% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740?GBq/μmol with a total synthesis time of ～40-min from EOB.     

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization

The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-³H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([³H]H₂DIDS) and by using SDS-PAGE. Assay of ³²Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C₃ counterpart and can be regarded as C₄ types.     

A deoxyribozyme that synthesizes 2',5'-branched RNA with any branch-site nucleotide

RNA molecules with internal 2′,5′-branches are intermediates in RNA splicing, and branched RNAs have recently been proposed as retrotransposition intermediates. A broadly applicable in vitro synthetic route to branched RNA that does not require self-splicing introns or spliceosomes would substantially improve our ability to study biochemical processes that involve branched RNA. We recently described 7S11, a deoxyribozyme that was identified by in vitro selection and has general RNA branch-forming ability. However, an important restriction for 7S11 is that the branch-site RNA nucleotide must be a purine (A or G), because a pyrimidine (U or C) is not tolerated. Here, we describe the compact 6CE8 deoxyribozyme (selected using a 20 nt random region) that synthesizes 2′,5′-branched RNA with any nucleotide at the branch site. The Mn²⁺-dependent branch-forming ligation reaction is between an internal branch-site 2′-hydroxyl nucleophile on one RNA substrate with a 5′-triphosphate on another RNA substrate. The preference for the branch-site nucleotide is U > C A > G, although all four nucleotides are tolerated with useful ligation rates. Nearly all other nucleotides elsewhere in both RNA substrates allow ligation activity, except that the sequence requirement for the RNA strand with the 5′-triphosphate is 5′-pppGA, with 5′-pppGAR (R = purine) preferred. These characteristics permit 6CE8 to prepare branched RNAs of immediate practical interest, such as the proposed branched intermediate of Ty1 retrotransposition. Because this branched RNA has two strands with identical sequence that emerge from the branch site, we developed strategies to control which of the two strands bind with the deoxyribozyme during the branch-forming reaction. The ability to synthesize the proposed branched RNA of Ty1 retrotransposition will allow us to explore this important biochemical pathway in greater detail.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

Clamping down on weak terminal base pairs: oligonucleotides with molecular caps as fidelity-enhancing elements at the 5'- and 3'-terminal residues

The base-pairing fidelity of oligonucleotides depends on the identity of the nucleobases involved and the position of matched or mismatched base pairs in the duplex. Nucleobases forming weak base pairs, as well as a terminal position favor mispairing. We have searched for 5′-appended acylamido caps that enhance the stability and base-pairing fidelity of oligonucleotides with a 5′-terminal 2′-deoxyadenosine residue using combinatorial synthesis and MALDI-monitored nuclease selections. This provided the residue of 4-(pyren-1-yl)butyric acid as a lead. Lead optimization gave (S)-N-(pyren-1-ylmethyl)pyrrolidine-3-phosphate as a cap that increases duplex stability and base-pairing fidelity. For the duplex of 5′-AGGTTGAC-3′ with its fully complementary target, this cap gives an increase in the UV melting point T_m of +10.9°C. The T_m is 6.3–8.3°C lower when a mismatched nucleobase faces the 5′-terminal dA residue. The optimized cap can be introduced via automated DNA synthesis. It was combined with an anthraquinone carboxylic acid residue as a cap for the 3′-terminal residue. A doubly capped dodecamer thus prepared gives a melting point decrease for double-terminal mismatches that is 5.7–5.9°C greater than that for the unmodified control duplex.