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Xiaolian Gao

Publications and source records attributed to Xiaolian Gao.

At least 19 recordsLinked to original sources
PubMed2006

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction.

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.

DNA↗
PubMed2006

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Amino Acid Sequence↗
PubMed2006

Another side of genomics: synthetic biology as a means for the exploitation of whole-genome sequence information.

The successful completion of the Human Genome Project and other sequencing projects opened the door for another quantum jump in science advancement. The most important public sequence databases are doubling in size every 18 months. By revealing the genetic program of many organisms, these efforts endow biologists with the ability to study the basic information of life in toto as an initial step toward a comprehensive understanding of the complexity of entire organisms. We review the area of synthetic biology, defined as the making and use of biosystems founded on the chemical synthesis of the coding DNA (and potentially RNA). The recent developments discussed here introduce a rich source of oligonucleotides to the field: in situ synthesised microarrays, which in fact represent nothing else but matrix nucleic acid synthesisers. With this new way of producing the oligonucleotides used in the making of synthetic genes in a very cost-effective manner, the field of synthetic biology can be expected to change dramatically in the next decade. Synthetic genes will then be the tools of choice to obtain any sequence at any time in any laboratory.

Base Sequence↗
PubMed2005

Nucleotide modification at the gamma-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase.

The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their gamma-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the gamma-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.

Binding Sites↗
PubMed2005

NMR structure and Mg2+ binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit.

Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G*A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037-1043 and 1112-1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson-Crick/Watson-Crick (cis W.C.) G*A conformation for the tandem (G*A)2 in the analog and a minimally perturbed helical duplex stem. Mg2+ titration studies imply that the cis W.C. geometry of the tandem (G*A)2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg2+ coordination.

Adenine↗
PubMed2005

IP-COSY, a totally in-phase and sensitive COSY experiment.

The IP-COSY experiment presented in this paper gives an in-phase spectral presentation in both the F(1) and F(2) dimensions by a combined use of a constant evolution time (CT) in t(1) and a symmetrical refocusing period before t(2). Compared with DQF-COSY and CT-COSY, IP-COSY further alleviates the effect of signal reduction due to a small ratio p (= J/linewidth), showing (1) improved lineshape and cross-peak definition and (2) especially enhancement in signals of the peaks of small active J coupling constant and the peaks of broader linewidth. A new strategy was adopted to eliminate or reduce effectively artifactual peaks by adding a 0.1-0.2 ms variation to the time delays of the CT period used for each scan of the FID in IP-COSY and CT-COSY. (3)J(H,H) coupling constants of larger than 4 Hz in the fingerprint region of peptides can be directly derived from the separation of doublets. IP-COSY cross peaks are stronger than those in DQF-COSY by 4-20-fold for tested peptides and oligonucleotides (MW < 8 kDa) with acquisition and processing parameters used in the work, and they are easier to identify than those in CT-COSY. The overall improvement in IP-COSY should make the detection/autodetection of the COSY cross peaks and the measurements of the various coupling constants more easily achieved, providing valuable information for the structure elucidation of peptides/small proteins and oligonucleotides.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed2005

The External RNA Controls Consortium: a progress report.

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

Animals↗
PubMed2004

Accurate multiplex gene synthesis from programmable DNA microchips.

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Escherichia coli↗
PubMed2004

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.

Base Sequence↗
PubMed2004

Gene2Oligo: oligonucleotide design for in vitro gene synthesis.

There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

Algorithms↗
PubMed2004

In situ synthesis of oligonucleotide microarrays.

This contribution presents a brief overall look of the methods for the preparation of various types of DNA microarrays and a thorough examination of the methods for in situ synthesis of oligonucleotide microarrays.

Biotechnology↗
PubMed2004

(3,2)D GFT-NMR experiments for fast data collection from proteins.

High throughput structure determination of proteins will contribute to the success of proteomics investigations. The G-Matrix Fourier Transformation NMR (GFT-NMR) method significantly shortens experimental time by reducing the number of the dimensions of data acquisition for isotopically labeled proteins (Kim, S. and Szyperski, T. (2003) J. Am. Chem. Soc. 125, 1385). We demonstrate herein a suite of ten 3D-->2D or (3,2)D GFT-NMR experiments using (13)C/(15)N-labeled ubiquitin. These experiments were completed within 18 hours, representing a 4- to 18-fold reduction in data acquisition time compared to the corresponding conventional 3D experiments. A subset of the GFT-NMR experiments, (3,2)D HNCO, HNCACB, HN(CO)CACB, and 2D (1)H-(15)N HSQC, which are necessary for backbone assignments, were carried out within 6 hours. To facilitate the analysis of the GFT-NMR spectra, we developed automated procedures for viewing and analyzing the GFT-NMR spectra. Our overall strategy allows (3,2)D GFT-NMR experiments to be readily performed and analyzed. Nevertheless, the increase in spectral overlap and the reduction in signal sensitivity in these fast NMR experiments presently limit their application to relatively small proteins.

Carbon Isotopes↗
PubMed2004

High density peptide microarrays. In situ synthesis and applications.

The technologies enabling the creation of large scale, miniaturized peptide or protein microarrays are emerging. The focuses of this review are the synthesis and applications of peptide and peptidomimetic microarrays, especially the light directed parallel synthesis of individually addressable high density peptide microarrays using a novel photogenerated reagent chemistry and digital photolithography (Gao et al., 1998, J. Am. Chem. Soc. 120, 12698; Pellois et al. 2002, Nat. Biotechnol. 20, 922). Concepts related to the synthesis are discussed, such as the reactions of photogenerated acids in the deprotection step of peptide synthesis or oligonucleotide synthesis, and the applications of high density peptide chips in antibody binding assays are discussed. Peptide chips provide versatile tools for probing antigen-antibody, protein-protein, peptide-ligand interactions and are basic components for miniaturization, automation, and system integration in research and clinical diagnosis applications.

Peptide Library↗
PubMed2004

TROSY-based correlation and NOE spectroscopy for NMR structural studies of large proteins.

Transverse relaxation-optimized spectroscopy (TROSY) is based on the fact that cross-correlation relaxation rates associated with the interferences between chemical shift anisotropy and dipole-dipole interactions can be dramatically reduced. TROSY selects these slowly relaxing components of 15N-1HN or 13C-1H antiphase coherences to significantly enhanced signal sensitivity and spectral resolution for large proteins (>30 kD). The basic principles and applications of three- and four-dimensional TROSY-based triple-resonance experiments and NOESY experiments for structure-function studies of proteins are discussed in this chapter. To make applications of these experiments easier, some of the experimental setups are also described.

Carbon Isotopes↗
PubMed2004

TROSY-based NMR experiments for the study of macromolecular dynamics and hydrogen bonding.

Transverse relaxation-optimized spectroscopy (TROSY)-based nuclear magnetic resonance (NMR) experiments can be exploited to obtain chemical shift assignment and values of J-coupling constants, residual dipolar couplings, and nuclear Overhauser effects (NOEs) for structural studies of proteins, as discussed in Chapter 5. Furthermore, the application of TROSY-based NMR experiments can be extended to the measurements of molecule dynamics, amide proton exchange rates, and hydrogen bonds. This chapter describes these experiments.

Hydrogen Bonding↗
PubMed2003

Glutamine cycling in isolated working rat heart.

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.

Adaptation, Physiological↗
PubMed2003

New complex of post-activated neocarzinostatin chromophore with DNA: bulge DNA binding from the minor groove.

Neocarzinostatin (NCS-chrom), a natural enediyne antitumor antibiotic, undergoes either thiol-dependent or thiol-independent activation, resulting in distinctly different DNA cleavage patterns. Structures of two different post-activated NCS-chrom complexes with DNA have been reported, revealing strikingly different binding modes that can be directly related to the specificity of DNA chain cleavage caused by NCS-chrom. The third structure described herein is based on recent studies demonstrating that glutathione (GSH) activated NCS-chrom efficiently cleaves DNA at specific single-base sites in sequences containing a putative single-base bulge. In this structure, the GSH post-activated NCS-chrom (NCSi-glu) binds to a decamer DNA, d(GCCAGAGAGC), from the minor groove. This binding triggers a conformational switch in DNA from a loose duplex in the free form to a single-strand, tightly folded hairpin containing a bulge adenosine embedded between a three base pair stem. The naphthoate aromatic moiety of NCSi-glu intercalates into a GG step flanked by the bulge site, and its substituent groups, the 2-N-methylfucosamine carbohydrate ring and the tetrahydroindacene, form a complementary minor groove binding surface, mostly interacting with the GCC strand in the duplex stem of DNA. The bulge site is stabilized by the interactions involving NCSi-glu naphthoate and GSH tripeptide. The positioning of NCSi-glu is such that only single-chain cleavage via hydrogen abstraction at the 5'-position of the third base C (which is opposite to the putative bulge base) in GCC is possible, explaining the observed single-base cleavage specificity. The reported structure of the NCSi-glu-bulge DNA complex reveals a third binding mode of the antibiotic and represents a new family of minor groove bulge DNA recognition structures. We predict analogue structures of NCSi-R (R = glu or other substituent groups) may be versatile probes for detecting the existence of various structures of nucleic acids. The NMR structure of this complex, in combination with the previously reported NCSi-gb-bulge DNA complex, offers models for specific recognition of DNA bulges of various sizes through binding to either the minor or the major groove and for single-chain cleavage of bulge DNA sequences.

Antibiotics, Antineoplastic↗