The analogue of thymidine triphosphate containing a methylene group in place of the 5' oxygen can serve as a substrate for reverse transcriptase.

The thymidine 5'-triphosphate analogue containing a methylene group in place of the 5' oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit beta-globin mRNA as the template together with an appropriate primer, the enzyme readily makes full-length DNA transcripts in which all thymidine 5' oxygen atoms have been replaced with methylene groups. In sequence analyses using the partial depurination procedure, the analogue DNA transcript produces electrophoretic gel patterns identical with those of the corresponding natural DNA transcript. Experiments on second strand synthesis using the four regular triphosphates show that the analogue DNA transcript, like the natural transcript, can serve as a template. The two DNA duplexes (natural/natural and analogue/natural) formed by these reactions produce similar electrophoretic cleavage patterns when treated with either of the endonucleases HaeIII and EcoRI. However, further studies on template properties indicate that, while the enzyme makes a full-length product when using the analogue substrate with a natural DNA strand as template, it appears unable to use the analogue transcript as template with the analogue triphosphate as substrate during second strand synthesis. Preliminary experiments have also been carried out with a DNA polymerase. No products are detected reactions using Taq polymerase with PCR protocols containing the analogue triphosphate as the only source of thymidine.

2'-Hydroxyl-protecting groups that are either photochemically labile or sensitive to fluoride ions.

Protected ribonucleotide monomers are more difficult to obtain than their 2'-deoxy counterparts because of the need to protect the 2'-hydroxy function. This unit describes the stepwise preparation of suitably 2'-protected ribonucleosides using two protecting groups: 2-nitrobenzyloxymethyl (NBOM) and tert-butyldimethylsilyl (TBDMS). In addition, details are given for protecting the 5'-hydroxyl and the nucleobase, yielding nucleosides that are easily converted to phosphoramidite or H-phosphonate derivatives for automated oligoribonucleotide synthesis.

Synthesis of oligoribonucleotides using the 2-nitrobenzyloxymethyl group for 2'-hydroxyl protection.

RNA can be rapidly synthesized using protected ribonucleoside phosphoramidite monomers that carry 2-nitrobenzyloxymethyl groups on their 2-hydroxyls. These protecting groups are removed from the oligoribonucleotide products by exposure to long-wave UV light.

Synthesis and properties of adenosine oligonucleotide analogues containing methylene groups in place of phosphodiester 5'-oxygens.

The ADP analogue in which the 5'-oxygen has been replaced by a methylene group can be prepared by condensing 5'-deoxy-5'-phosphonomethyladenosine with inorganic phosphate. This analogue readily polymerizes onto the primer A-A in the presence of the enzyme polynucleotide phosphorylase and either Mg2+ or Mn2+. The initial products are of the form A-A(-cA)n-cA (where "-" and "-c" stand for the normal phosphodiester linkage and the linkage in which the 5'-oxygen is replaced with the methylene group, respectively). Treatment of these with alkali yields adenosine 2'(3')-phosphate and the series (A(-cA)n-cA containing only phosphonomethylene linkages. The decamer A(-cA)8-cA interacts with two molecules of U(-U)8-U to form a triple-standard structure that has a stability similar to that exhibited by the analogous complex formed from A(-A)8-A and U(-U)8-U. This property, along with the resistance of these oligomer analogues toward nucleases that cleave phosphodiester linkages between the phosphorus and the 5'-oxygen, should provide a strong rationale for application of phosphonomethylene linkages in schemes for therapeutic drug design that use the antisense strategy.

Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester.

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.

Crystallographic structure of an RNA helix: [U(UA)6A]2.

The crystallographic structure of the synthetic oligoribonucleotide, U(UA)6A, has been solved at 2.25 A resolution. The crystallographic refinement permitted the identification of 91 solvent molecules, with a final agreement factor of 13%. The molecule is a dimer of 14 base-pairs and shows the typical features of an A-type helix. However, the presence of two kinks causes a divergence from a straight helix. The observed deformation, which is stabilized by a few hydrogen bonds in the crystal packing, could be due to the relatively high (35 degrees C) temperature of crystallization. The complete analysis of the structure is presented. It includes the stacking geometries, the backbone conformation and the solvation.

High resolution structure of the RNA duplex [U(U-A)6A]2.

RNA is involved in many biological functions, ranging from information storage and transfer to the catalysis of reactions involving both nucleic acids and proteins. Previous crystallographic studies on RNA oligomeric chains provide only averaged structures or information limited in resolution. The oligomer [U(U-A)6A]2 was chosen for the study of protein-RNA interactions in viruses. Its size and base composition mimic portions of the genomic RNA in alfalfa mosaic virus that bind to the amino terminus of the viral subunit. The actual sequence was designed to guarantee the formation of a single species of duplex and to facilitate the production of the pure oligomer in large quantities. The molecular structure, derived from the 2.25 A resolution X-ray diffraction data, allows the most detailed analysis of an A-RNA helix reported to date. Two kinks are observed that divide the duplex into three blocks, each close to a canonical A-helical conformation. A few intermolecular hydrogen bonds involving 2'-hydroxyl groups stabilize this peculiar conformation of the RNA, which may be related to the temperature used for the crystallization (35 degrees C). The structure demonstrates both the plasticity of the RNA molecule and the role of the 2'-hydroxyl groups in intermolecular interactions.

Formation of MboII vectors and cassettes using asymmetric MboII linkers.

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.

Use of ribonucleosides as protecting groups in synthesis of polynucleotides with phosphorylated terminals.

Two new protected 5'-ribonucleotides, 2'3'-O-bis(4,4'-dimethoxytrityl)uridine 5'-(4-chlorophenyl phosphate) and 2',3'-O-(methoxymethylene)uridine 5'-(4-chlorophenyl phosphate), form the basis of a chemical procedure for phosphorylating the 5'-ends of DNA fragments synthesized by the phosphotriester approach. Condensation of either of these mononucleotide units with the free 5'-hydroxyl of an otherwise fully protected oligomer results in high-yield formation of a 5'-5' triester linkage. Subsequently, the terminal ribonucleotide of the deprotected product rU5'-5'd(N-Nn-N) can be cleaved by periodate oxidation of its 2',3'-cis-hydroxyl system followed by beta-elimination, leaving its phosphate attached to the 5'-hydroxyl group of the oligodeoxyribonucleotide. This procedure together with a tactic employing a 2',3'-O-acylribonucleotide residue at the 3'-terminus of the chain allows the synthesis of oligomers possessing monophosphate groups at either end or both ends. Furthermore, oligonucleotide intermediates possessing a 5'-5'-linked uridine terminal are shown to have a special application as acceptors in RNA ligase reactions, where the presence of the ribonucleoside cap on the 5'-phosphate limits ligation specifically to the 3'-ends of the oligomers. Removal of the uridine residues to expose free 5'-phosphates would then enable the products to participate as donors in further elongation reactions.

Imino proton assignments in the proton nuclear magnetic resonance spectrum of the lambda phage OR3 deoxyribonucleic acid fragment.

The 17 base pair duplex d(TATCACCGCAAGGGATAp) . d(TATCCCTTGCGGTGATAp) corresponding to the OR3 operator site of lambda phage has been synthesized and studied by 1H nuclear magnetic resonance spectroscopy at 470 MHz. The 13 imino proton resonances observed at 20 degrees C have been assigned to specific base pairs at positions 3-15 on the basis of nuclear Overhauser effect measurements and studies of the temperature dependence of peak intensities. Resonances from the A-T base pairs at positions 1, 2, 16, and 17 are assumed to be absent from the spectrum because of terminal fraying. Resonance from many of the base pairs suggested by Ohlendorf et al. [Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., & Matthews, B. W. (1982) Nature (London) 298, 718-723] to be involved in specific binding of the lambda phage cro repressor are well resolved.

Ribonucleoside and ribonucleotide derivatives in polynucleotide synthesis.

Suitably protected ribonucleotide and ribonucleoside derivatives have been employed as versatile intermediates in both oligoribo- and oligodeoxyribonucleotide synthesis by the phosphotriester method. Thus, the barium salt of 5'-O-dimethoxytrityl-2'-O-(o-nitrobenzyl) uridine-3' p-chlorophenyl-phosphate was used to prepare U-U-U as one example of a procedure that has general applicability in the construction of oligoribonucleotides. In addition, ribonucleosides like N4,O2',O3'-tribenzoylcytidine, and ribonucleotides such as the 2',3'-O-bis(dimethoxytrityl) and 2',3'-O-methoxymethylidene derivatives of uridine-5' p-chlorophenyl phosphate, provide a convenient means of introducing 3'- and/or 5'-terminal phosphate residues into oligonucleotides at the phosphotriester level.

The use of barium salts of protected deoxyribonucleoside-3' p-chlorophenyl phosphates for construction of oligonucleotides by the phosphotriester method: high-yield synthesis of dinucleotide blocks.

A new experimental approach to the synthesis of polydeoxyribonucleotides via the phosphotriester method involves construction of oligonucleotide blocks by direct use of the easily prepared barium salts of O5',N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates as the key monomers in condensation reactions. The procedure has been demonstrated by the rapid synthesis in high yield and purity of all sixteen fuly protected dinucleotides (Formula: see text) (where dN' = dT, dbzC, dbzA, or dibG; (Formula: see text) This set of molecules constitutes a "syllabary" for the preparation of defined sequence oligonucleotides.

Protected deoxyribonucleoside-3' aryl phosphodiesters as key intermediates in polynucleotide synthesis. Construction of an icosanucleotide analogous to the sequence at the ends of Rous sarcoma virus 35S RNA.

Several modifications have been incorporated into the phosphotriester strategy for chemical synthesis of oligodeoxyribonucleotides. These include high-yield methods of preparation and isolation of O5', N-protected deoxyribonucleoside-3' p-chlorophenyl phosphates which serve as key intermediates, and the elimination of some superfluous manipulation and purification steps commonly used in the process of synthesizing oligonucleotide blocks. In addition, two new arylsulfonyl nitroimidazole derivatives have been prepared and found to be highly effective agents for internucleotide bond formation. These techniques have been applied in construction of the iconsamer d(G-C-C-A-T-T-T-T-A-C-C-A-T-T-C-A-C-C-A)-rC, equivalent to a ribonucleotide sequence located at both the 5' and 3' ends of Rous sarcoma virus 35S RNA.

Changes in thymus size observed radiographically during the course of spontaneous lymphoma in the AKR/J mouse.

Previous means of diagnosing AKR lymphoma only detected mice in fairly advanced stages of the disease. Since the first organ to be affected is usually the thymus, a study was made of the feasibility of diagnosing the disease and following its course by observing changes in thymus size radiographically. Rapid exposure of unanesthetized AKR/J mice to relatively soft X rays provided adequate radiographs with negligible doses to the mice (3-4 mrad). Planimeter measurements made of the cross-sectional area around the heart and thymus in side view radiographs of the individually housed mice provided a quantitative and reproducible measure of the thymus size for thymuses greater than 100 mg. When these measurements were added to the palpation scores of spleen and lymph nodes, all leukemic mice were detected. Thymus size increases were not detected until late in the disease, and decrease in thymus size was seen in approximately half of the mice prior to death. This precluded direct correlation of size to time of death. There was no relationship between the preterminal decrease in thymus size and increasing or decreasing blood leukocyte counts or leukocyte size.

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.