Conformation of N-(purin-6ylcarbamoyl) glycine, a hypermodified base in tRNA

The crystal structure of the potassium salt of N-(purin-6ylcarbamoyl) glycine was determined from three-dimensional X-ray diffraction data. The N⁶-substituent is distal ($\text{trans}$) to the imidazole ring, forming an intramolecular hydrogen bond N(glycine) -H---N(1)adenine. This conformation of the N⁶-substituent is typical of ureidopurines, and blocks the two sites N⁶-H and N¹ of adenine that are normally utilized for complementary base-pairing in the double helical regions of nucleic acids; the internal hydrogen bonding further enhances the shielding of N¹. This blocking of N⁶-H and N¹ may be important in enhancing the single stranded conformation of the anticodon loop of tRNA and in preventing the modified adenosine adjacent to the anticodon from taking part directly in codon-anticodon interaction through the complementary base pairing.     

Conformation of N4-acetylcytidine,a modified nucleoside of tRNA,and stereochemistry of codon-anticodon interaction

The crystal structure of N⁴-acetylcytidine (ac⁴C) a modified nucleoside of tRNA, has been determined from three-dimensional x-ray diffractometer data. The N⁴-substituent is proximal to C(5), quite contrary to expectations from solution studies of N⁴-methylcytosine. This orientation of the N⁴-substituent will not block Watson-Crick base pairing for reading the third codon by tRNA_M^Met, and hence the discriminatory function suggested for ac⁴C might arise due to non-standard conformation of the polynucleotide backbone of the anticodon around the Wobble base. A common characteristic of the modified nucleosides that occur at the Wobble position is their inability to shield the Watson-Crick base pairing sites; this is quite consistent with the necessity for reading the third base of the codon. This is in sharp contrast to the modifications of the nucleosides adjacent to the 3′-end of anticodons, all of which prevent Watson-Crick base pairing.     

Theoretical study on the binding mechanism between N6-methyladenine and natural DNA bases

N6-methyladenine (m⁶A) is a rare base naturally occurring in DNA. It is different from the base adenine due to its N-CH₃. Therefore, the base not only pairs with thymine, but also with other DNA bases (cytosine, adenine and guanine). In this work, Møller-Plesset second-order (MP2) method has been used to investigate the binding mechanism between m⁶A and natural DNA bases in gas phase and in aqueous solution. The results show that N-CH₃ changed the way of N6-methyladenine binding to natural DNA bases. The binding style significantly influences the stability of base pairs. The trans-m⁶A:G and trans-m⁶A:C conformers are the most stable among all the base pairs. The existence of solvent can remarkably reduce the stability of the base pairs, and the DNA bases prefer pairing with trans-m⁶A to cis-m⁶A. Besides, the properties of these hydrogen bonds have been analyzed by atom in molecules (AIM) theory, natural bond orbital (NBO) analysis and Wiberg bond indexes (WBI). In addition, pairing with m⁶A decreases the binding energies compared to the normal Watson-Crick base pairs, it may explain the instability of the N6 site methylated DNA in theory.

Incorporation of 3H-adenine into free cytokinins by cytokinin-autonomous tobacco callus tissue

Cytokinin-autonomous tobacco callus was incubated in defined mineral medium containing ³H-adenine for 60 minutes. Radioactivity was incorporated into the four predominant free cytokinins, ribosyl-$\text{trans}$-zeatin, $\text{trans}$-zeatin, N⁶-(Δ²-isopentenyl) adenosine and N⁶-(Δ²-isopentenyl) adenine. The bases were more abundant than their respective ribosides, N⁶-(Δ²-isopentenyl) adenine being the most abundant cytokinin. No discrete peaks of radioactivity could be detected on the HPLC column eluate corresponding to the elution volumes of $\text{cis}$-zeatin and ribosyl-$\text{cis}$-zeatin.     

Molecular structure of 2,2′-anhydro-1-β-D-arabinofuranosyl cytosine hydrochloride (cyclo ARA-C): A highly rigid nucleoside

The molecular structure of cyclo ara-C hydrochloride has been determined by x-ray diffraction methods. The ether linkage between the base and sugar moieties severely restricts the conformation about the glycosyl bond and the mode of sugar puckering. The glycosyl torsion angle (X_CN =299°) lies in a region outside the $\text{anti}\text{and}\text{syn}$ ranges found for the β-nucleosides. The arabinose ring exhibits C(4′)-endo (⁴E) mode of puckering, with a pseudorotation phase angle P of 233°. The positive charge on the base apparently stabilizes the $\text{gauche}\text{-}\text{gauche}$ conformation of the C(5′)-O(5′) bond despite the short contacts between O(5′) and C(2) and N(1) of the base.     

Conformational Preferences of Modified Nucleic Acid Bases N6-Methyl-N6-(N-Threonylcarbonyl) Adenine and 2-Methylthio-N6-(N-Threonylcarbonyl) Adenine by the Quantum Chemical PCILO Calculations

Abstract

Conformational preferences of the hypermodified nucleic acid bases N⁶-methyl-N⁶-(N- threonylcarbonyl) Adenine, m⁶ tc⁶ Ade, and 2-methylthio-N⁶-(N-threonylcarbonyl) Adenine, mS² tc⁶ Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m⁶ tc⁶ Ade and mS² tc⁶ Ade alike the threonylcar- bonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N⁶-(N-threonylcarbonyl) Adenine, tc⁶ Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc⁶ Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(l) of the adenine base.

The N⁶-methyl protons, in m⁶ tc⁶ Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS² tc⁶ Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N⁶-(Δ²-isopentenyl) Adenine, mS² i⁶ Ade, where the 2-methylthio group orients trans to the C(2)- N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Crystal structure of an inhibitor and a model for inhibition of replicative DNA synthesis

6-(p-Hydroxyphenylhydrazino)-uracil is an antimicrobial agent that selectively blocks replicative DNA synthesis in Bacillus subtilis by inhibiting DNA polymerase III. The drug crystallizes as a monoclinic monohydrate, space group $\text{C2}\text{c}$, with $\text{a = 23.920(6)}\text{A}\text{̊}\text{, b = 5.587(3)}\text{A}\text{̊}\text{, c = 17.466(5)}\text{A}\text{̊}\text{, β = 101.45(8) °}$, and eight hydrated molecules per cell. Three-dimensional X-ray diffraction data were collected. The structure was solved by Patterson methods and refined to an R value of 6.8% for the 1651 data. The geometry of the uracil ring is unusual. The bond distances suggest that a resonance form involving a positively charged hydrazino nitrogen and a negatively charged carbonyl oxygen, O(4), makes a large contribution to the valence bond structure of this compound. The exocyclic C(6)N bond is short (1.335 Å), the C(6)C(5) bond distance is 1.371 Å, which is longer than in uracil, and the C(5)C(4) distance (1.396 Å) is short. The uracil ring, the linked hydrazino nitrogen, and the hydrogen on this nitrogen are in the same plane. Each uracil group is hydrogen bonded to a nearly coplanar uracil across a center of symmetry. The water molecule is also near the plane of these paired bases and forms a hydrogen bond with the uracil-linked hydrazino NH group. This paired base arrangement and the restricted rotation about the exocyclic C(6)N link that constrains the hydrazino NH group to lie near the uracil plane suggest a model for the interaction of the drug with template-primer DNA. The drug acts when cytosine is the base to be copied in the template strand, and the drug is competitive with dGTP. Both cytosine and guanine can be accommodated with little distortion of the crystal structure geometry in a manner compatible with the known geometry of DNA. The structural and biochemical aspects of the model for drug action are discussed.     

Molecular mechanical studies of base-pair opening in d(CGCGC):d(GCGCG), dG5·dC5, d(TATAT):d(ATATA), and dA5·dT5 in the B and Z forms of DNA

Molecular mechanical energy refinement of double-helical pentanucleotide tetra-phosphates, d(CGCGC):d(GCGCG), dG₅·dC₅, d(TATAT):d(ATATA), and dA₅ ·dT₅ geometries, are presented in order to examine the energy required to open the Nl(purine) …? N3(pyrimidine) distance (base-pair opening) of a Watson-Crick base pair from its normal value of 3 Å to a value of 6 Å. The structural consequences of forcing base-pair opening is sequence dependent. For both dA₅ ·dT₅ and d(TATAT):d(ATATA), forcing the Nl (AdeKN3 (Thy) distance of the central base pair to a value of 6 Å slides the bases perpendicular to the helix axis forming a low-energy non-Watson-Crick base pair having an adenine amine hydrogen …? thymine carbonyl oxygen hydrogen bond. The two GC sequences behave differently from both AT sequences and differently from each other. Forcing the Nl(Gua) …? N3(Cyt) distance to 6 Å leads to unconventional structures in which hydrogen bonds are formed between the separated bases and the bases above or below them. These structures appear to be trapped in true local minima 6–10 kcal/mol higher in energy than the Watson-Crick structures. Preliminary simulations on d(CGCGC):d(GCGCG) in the Z geometry suggest the reason the Z form may be more refractory to proton exchange than the B form, consistent with experimental observations.     

Ordered transcription of RNA tumor virus genomes.

The crystal structure of sodium adenylyl-3′,5′-uridine (ApU) hexahydrate has been determined by X-ray diffraction procedures and refined to an R factor of 0.057. ApU crystallizes with two molecules per asymmetric unit in a monoclinic unit cell, space group P2₁, with cell dimensions: $\text{a = 18.025, b = 17.501, c = 9.677}\text{A}\text{?}\text{and}\text{β = 99.45 °}$. The two independent molecules of ApU form a small segment of right-handed antiparallel double-helical RNA in the crystal, with Watson-Crick base-pairing between adenine and uracil. This is the first time that this Watson-Crick base-pair has been seen unambiguously at atomic resolution and it is also the first time that a nucleic acid fragment with double-helical symmetry has been seen at atomic resolution. The distance between the C1′ atoma of the adenine-uracil base-pair is slightly shorter than the analogous distance seen in guanine-cytosine base-pairs. The bases in each strand are heavily stacked. One sodium cation binds to the phosphates, as expected; however, the other sodium cation binds on the dyad axis in the minor groove of the double helix. It is co-ordinated directly to the two uracil carbonyl groups which protrude into the minor groove and is shielded from the nearest phosphates by a shell of water. This binding appears to be sequence-specific for ApU. One of the adenines also forms a pair of hydrogen bonds to a nearby ribose, utilizing N6 and N7. The 12 water molecules per double-helical fragment are all part of the first co-ordination shell. The ions and the symmetry of the double-helical fragment are the major organizing elements of the solvent region.     

Conformation of acetylaminofluorene and aminofluorene modified guanosine and guanosine derivatives

Acetylaminofluorene and aminofluorene modified Guo, GMP, d(GpA) and d(ApG) have been studied by circular dichroism and ¹H nuclear magnetic resonance. Aminofluorene modified Guo is preferentially in the $\text{anti}$ conformation and acetylaminofluorene modified Guo in the $\text{syn}$ conformation. It is proposed that the $\text{anti}$ conformation of aminofluorene modified Guo is stabilized by an intra molecular hydrogen bond between the NH group of aminofluorene residue and the 5′-OH group of the sugar. The results on the modified dinucleoside monophosphates are analyzed according to this hypothesis.     

Structural Features and Hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a Double Helix Containing Two G.A Mispairs

Abstract

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5Å resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-l and 0–6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Analysis of the inhibitor binding to the nucleoside site of phosphorylase b

The binding of inhibitors to site I of rabbit muscle phsphorylase $\text{b}$ has beenstudied kinetically and thermodynamically for caffeine, adenine and adenosine. The effect of ligands on the tertiary structure has been investigated by studying the protection against 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) titration of the slow-reacting sulphydryl groups of the enzyme. Calorimetric and cysteinyl protection data taken together suggest that these inhibitors bind to both sites N and I even under conditions of saturation by glucose. Calorimetric results show that inhibitor binding to sites I and N at 25°C is driven enthalpically, although both $\text{Δ}\text{H}$ and $\text{Δ}\text{S}$ of interaction are significant. We conclude that attractive dispersion forces ought to be the main ones responsible for inhibitor binding to site I. AMP-activated phosphorylase $\text{b}$ is inhibited by both caffeine and adenine by cooperative and exclusive binding to the inactive $\text{T}$ conformation. The binding of the substrate (phosphate) and AMP when adenine is present was found to be exlusive to the active $\text{R}$ conformation, whereas non-exclusive binding of the activator was observed when caffeine was added.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

The crystal structure of the octamer [r(guauaca)dC]2 with six Watson-Crick base-pairs and two 3' overhang residues

The crystal structure of an alternating RNA octamer, r(guauaca)dC (RNA bases are in lower case while the only DNA base is in upper case), with two 3' overhang residues one of them a terminal deoxycytosine and the other a ribose adenine, has been determined at 2.2 A resolution. The refined structure has an Rwork 18.6% and Rfree 26.8%. There are two independent duplexes (molecules I and II) in the asymmetric unit cell, a = 24.95, b = 45.25 and c = 73.67 A, with space group P2(1)2(1)2(1). Instead of forming a blunt end duplex with two a+.c mispairs and six Watson-Crick base-pairs, the strands in the duplex slide towards the 3' direction forming a two-base overhang (radC) and a six Watson-Crick base-paired duplex. The duplexes are bent (molecule I, 20 degrees; molecule II, 25 degrees) and stack head-to-head to form a right-handed superhelix. The overhang residues are looped out and the penultimate adenines of the two residues at the top end (A15) are anti and at the bottom (A7) end are syn. The syn adenine bases form minor groove A*(G.C) base triples with C8-H...N2 hydrogen bonds. The anti adenine in molecule II also forms a triple and a different C2-H...N3 hydrogen bond, while the other anti adenine in molecule I does not, it stacks on the looped out overhang base dC. The 3' terminal deoxycytosines form two stacked hemiprotonated trans d(C.C)+ base-pairs and the pseudo dyad related molecules form four consecutive deoxyribose and ribose zipper hydrogen bonds in the minor groove.     

6-Azauridine, a nucleoside with unusual ribose conformation. The molecular and crystal structure

The cytostatic analogue ribo-6-azauridine crystallizes in the orthorhombic space group P2₁2₁2₁ with eight molecules per unit cell of dimensions $\text{a = 20.230, b = 7.709, c = 12.863}\text{A}\text{?}$. A trial structure was obtained by direct methods. Least-squares refinement of co-ordinates and anisotropic thermal parameters based on 1998 reflections measured on a four-circle diffractometer led to a discrepancy index R = 4.0%. Like uridine, 6-azauridine has the anti conformation about the glycosidic bond and a C(3′)-endo sugar pucker. Unlike uridine, it exhibits a close approach of N(6) to C(2′) at only 2.814 and 2.844 Å in the two independent molecules, and a C(5′)(5′) bond that is gauche to C(4′)O(1′) but trans to C(4′)C(3′); this conformation about a C(4′)C(5′) bond has never been observed before for C(3′)-endo puckered riboses in the crystalline state. The crystal structure displays a pseudo-A face centering and very similar conformational parameters for the two independent molecules. Every OH and NH group in the structure serves as a proton donor in a hydrogen bond, including an unusual N(3)—H(3) … O(1′) link. Molecular orbital calculations by the extended Hückel method indicate that from uridine to 6-azauridine the net charge changes sign at ring positions 5 and 6 and disappears at 1.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Water structure in a protein crystal: rubredoxin at 1.2 A resolution

The model for rubredoxin based on X-ray diffraction data has been extensively refined with a 1.2 Å resolution data set. Water oxygen atoms were deleted from the model if B exceeded 50 Ų and occupancy was less than 0.3 eÅ^?3. The final water model consists of 127 sites with B values ranging from 15 to $\text{6}\text{?}$0 Ų and occupancies from unity down to 0.3, the most tightly bound water oxygen atoms being hydrogen bonded to two or more main-chain nitrogen or oxygen atoms. The water forms extensive hydrogen bond networks bridging the crevices on the molecular surfaces and between adjacent molecules. The minimum distances of the water sites from the protein surface are distributed about two distinct maxima, the major one at 2.5 to 3 Å and a minor one at 4 to 4.5 Å. Beyond $\text{5}\text{?}$ to 6 Å from the protein surface, the discrete water merges into the aqueous continuum.     

The crystal and molecular structure of 8-methyladenosine 3'-monophosphate dihydrate.

8-Methyladenosine 3'-monophosphate dihydrate was synthesized and crystallized in the monoclinic space group P21 with the unit cell dimensions: a = 9.095(2) A, b = 16.750(3) A, c = 5.405(2) A and beta = 97.61(3) degrees. The structure was determined by the application of the heavy atom method and refined to give a final R factor of 0.047. The pertinent conformations are as follows: the syn conformation about the glycosyl bond (chiCN = 216.8 degrees), the C(2')-endo sugar puckering with the displacement of 0.55 A; and the gauche-gauche conformation about the C(4')-C(5') bond capable of forming an intramolecular hydrogen bonding between N(3) of adenine base and O(5') of the hydroxymethylene group on the ribose. The molecule exists in the zwitterionic form with the N(1) of the adenine base protonated by a phosphate proton and is stabilized by three-dimensional networks of hydrogen bonding through the crystalline water molecules or directly between the adjacent nucleotide molecules; no base stacking was observed.     

Cyclopenta(f)isoquinoline derivatives designed to bind specifically to native deoxyribonucleic acid. 3. Interaction of 6-carbamylmethyl-8-methyl-7H-cyclopenta(f)isoquinolin-3(2H)-one with deoxyribonucleic acids and polydeoxyribonucleotides

By the use of space-filling models, a novel compound, 6-carbamylmethyl-8-methyl-7$\text{H}$-cyclopenta[f]isoquinolin-3(2$\text{H}$)-one ($\text{1}$) was devised which would be expected to hydrogen bond specifically to GC pairs in the major groove of the double helix such that (i) the amino group of the cytosine molecule donates a hydrogen bond to the C-3 carbonyl of the isoquinoline moiety and (ii) the amide proton of the side chain donates a hydrogen bond to the N-7 of guanine. From difference spectra studies it was found that $\text{1}$ binds to native calf thymus DNA better than to denatured DNA; $\text{1}$ inhibited RNA synthesis by a DNA-dependent RNA polymerase; and equilibrium dialysis experiments revealed that $\text{1}$ binds to poly(dG).poly(dC), whereas no such binding to poly(dA).poly(dT) was observed.