Biomedical subjects

C Cheong

Publications and source records attributed to C Cheong.

9 recordsLinked to original sources

PubMed1992

Solution structure of an unusually stable RNA tetraplex containing G- and U-quartet structures.

A model for the solution structure of an RNA tetraplex, (rUGGGGU)4, has been obtained by two-dimensional NMR spectroscopy and molecular dynamics. The molecule is parallel stranded and Hoogsteen base-paired in 50 mM KCl, and it is so stable that three of its six imino protons have exchange half-lives measured in days at 40 degrees C. The tetraplex is stabilized by base stacking and by the hydrogen bonds in four G quartets and at least one U quartet. This is the first indication of the existence of U-quartet structures of which we are aware.

Hydrogen Bonding↗

PubMed1991

Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs.

The crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the middle of the sequence did not form an internal loop, but rather a highly regular double-helix incorporating the non-Watson-Crick base pairs, G.U and U.C. This is the first direct observation of a U.C (or T.C) base pair in a crystal structure. The U.C pairs each form only a single base-base hydrogen bond, but are stabilized by a water molecule which bridges between the ring nitrogens and by four waters in the major groove which link the bases and phosphates. The lack of distortion introduced in the double helix by the U.C mismatch may explain its low efficiency of repair in DNA. The G.U wobble pair is also stabilized by a minor-groove water which bridges between the unpaired guanine amino and the ribose hydroxyl of the uracil. This structure emphasizes the importance of specific hydrogen bonding between not only the nucleotide bases, but also the ribose hydroxyls, phosphate oxygens and tightly bound waters in stabilization of the intramolecular and intermolecular structures of double helical RNA.

Base Sequence↗

PubMed1991

Tetramerization of an RNA oligonucleotide containing a GGGG sequence.

Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.

Base Sequence↗

PubMed1991

Structure of an unusually stable RNA hairpin.

The structure of a very common RNA hairpin, 5'GGAC(UUCG)GUCC, has been determined in solution by NMR spectroscopy. The loop sequence, UUCG, occurs exceptionally often in ribosomal and other RNAs, and may serve as a nucleation site for RNA folding and as a protein recognition site. Reverse transcriptase cannot read through this loop, although it normally transcribes RNA secondary structure motifs. A hairpin with that loop displays unusually high thermodynamic stability; its stability decreases when conserved nucleotides are mutated. The three-dimensional structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR using distance geometry, followed by restrained energy minimization. The structure was well-defined despite the conservative use of interproton distances, by constraining the backbone conformation by means of scalar coupling measurements. A mismatch G.U base pair, with syn-guanosine, closes the stem. This hairpin has a loop of only two nucleotides; both adopt C2'-endo sugar pucker. A sharp turn in the phosphodiester backbone is stabilized by a specific cytosine-phosphate contact, probably a hydrogen bond, and by stacking of the cytosine nucleotide on the G.U base pair. The structural features of the loop can explain the unusual thermodynamic stability of this hairpin and its sensitivity to mutations of loop nucleotides.

Base Sequence↗

PubMed1990

Solution structure of an unusually stable RNA hairpin, 5'GGAC(UUCG)GUCC.

Hairpin loops are important structural elements of RNA, helping to define the three-dimensional structure of large RNAs and providing potential nucleation sites for RNA folding and interaction with other nucleic acids and proteins. Little, however, is known about the conformation of RNA hairpins, most of what we know coming from transfer RNA crystal structures and from studies of DNA hairpins. We report here the determination of the structure of a very stable and common RNA hairpin, 5'GGAC(UUCG)GUCC (loop nucleotides in parenthesis), by NMR spectroscopy. The sequence C(UUCG)G occurs very often in RNA and may be a nucleation site for RNA folding and a protein-binding site. A high-resolution structure for the hairpin was derived from interproton distances and scalar coupling constants determined by NMR. The loop is stabilized by a G.U base pair, with guanine in the syn conformation, a cytosine-phosphate contact and extensive base stacking. These findings and other structural features of the loop can explain the unusual stability of the hairpin and suggest why reverse transcriptase cannot read through the loop, although it can transcribe through other kinds of RNA secondary structure.

Base Composition↗

PubMed1989

Computerized schedules--one solution to variable workstyles.

A computerized scheduling program was developed to provide hospital anaesthetic coverage and yet still satisfy the complex workstyles of 30 anaesthetists. Hospital commitments required ten anaesthetists available each weekday and four each weekend day for after-hours coverage of the intensive care units and operating rooms. The workstyles included part-time arrangements, limited calls by some individuals, and calls restricted to certain anaesthetists with specific areas of expertise. Rules were defined to limit the proximity of late calls to avoid fatigue and computer software was developed. For each scheduling period, the program assists in making daily call assignments based on each anaesthetist's availability and a priority ranking system. It is flexible enough to allow personal preferences. The number and distribution of all calls scheduled are counted. When the assignments are completed, differences are reconciled. For two years, this program has proved superior to previous manual systems for scheduling this group of anaesthetists with variable workstyles.

Anesthesia Department, Hospital↗

PubMed1988

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.

2-Aminopurine↗

PubMed1988

Comparison between DNA melting thermodynamics and DNA polymerase fidelity.

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.

Base Sequence↗

PubMed1986

Vacuum ultraviolet circular dichroism as an indicator of helical handedness in nucleic acids.

Calculated circular dichroism spectra are presented for double-stranded polynucleotides of regular sequences in A-RNA, A-DNA, B-DNA, and Z-DNA conformations. Quantum mechanical matrix method calculations were carried out in the near and vacuum ultraviolet regions. In the near UV, the calculated spectra agreed qualitatively with the measured spectra. However in the far and vacuum UV, the calculated CD compared nearly quantitatively with the experimental spectra. The calculations show that the sign of the CD in the vacuum UV, in contrast to that in the near UV, can be correlated with the handedness of the helix.

Adenine↗