Gel-based oligonucleotide microarray approach to analyze protein-ssDNA binding specificity

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5'-{N}N(1)N(2)N(3)N(4)N(5)N(6){N}-3' consisted of a fixed hexamer motif N(1)N(2)N(3)N(4)N(5)N(6) in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase-ssDNA complexes with the temperature increasing from 0 degrees C to 50 degrees C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5'-NNG(A/T/C)GNN-3' with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.     

The 3'-terminal hexamer sequence of classical swine fever virus RNA plays a role in negatively regulating the IRES-mediated translation

The 3' untranslated region (UTR) is usually involved in the switch of the translation and replication for a positive-sense RNA virus. To understand the 3' UTR involved in an internal ribosome entry site (IRES)-mediated translation in Classical swine fever virus (CSFV), we first confirmed the predicted secondary structure (designated as SLI, SLII, SLIII, and SLIV) by enzymatic probing. Using a reporter assay in which the luciferase expression is under the control of CSFV 5' and 3' UTRs, we found that the 3' UTR harbors the positive and negative regulatory elements for translational control. Unlike other stem loops, SLI acts as a repressor for expression of the reporter gene. The negative cis-acting element in SLI is further mapped to the very 3'-end hexamer CGGCCC sequence. Further, the CSFV IRES-mediated translation can be enhanced by the heterologous 3'-ends such as the poly(A) or the 3' UTR of Hepatitis C virus (HCV). Interestingly, such an enhancement was repressed by flanking this hexamer to the end of poly(A) or HCV 3' UTR. After sequence comparison and alignment, we have found that this hexamer sequence could hypothetically base pair with the sequence in the IRES IIId1, the 40 S ribosomal subunit binding site for the translational initiation, located at the 5' UTR. In conclusion, we have found that the 3'-end terminal sequence can play a role in regulating the translation of CSFV.     

Infrared spectral studies of the non regularly alternating purine-pyrimidine hexamers d(m5CGGCM5CG), d(CBr8GGCCBr8G) and d(CGCGGC)

The oligonucleotides d(m5CGGCm5CG), d(CBr8GGCCBr8G) and d(CGCGGC) have been prepared and studied by infrared spectroscopy. The three sequences contain two GC pairs which are out of purine-pyrimidine alternation with the rest of the sequence. From the IR data of the d(m5CGGCm5CG) hexamer, it is shown that all of the dG residues adopt a syn conformation. The marker IR bands for the C3' endo syn conformation are at 1410, 1354, 1320 and 925 cm-1 whereas those for the C2' endo anti conformation at 1420, 1374 and 890 cm-1 are clearly absent. This result implies that the two adjacent guanines of the d(m5CGGCm5CG) sequence are in syn conformation. It is suggested that duplex formation occurs in d(CGCGGC) films and that all of the guanines are in syn conformation. In contrast, the central non-brominated guanine of the d(CBr8GGCCBr8G) hexamer is found in anti conformation, as expected in a Z type structure of the non-alternating region.     

A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene.

We identified two thyroid hormone response elements (TREs) in the 2.5-kb, 5'-flanking region of the human gene encoding type 1 iodothyronine deiodinase (hdio1), an enzyme which catalyses the activation of thyroxine to 3,5,3'-triiodothyronine (T3). Both TREs contribute equally to T3 induction of the homologous promoter in transient expression assays. The proximal TRE (TRE1), which is located at bp -100, has an unusual structure, a direct repeat of the octamer YYRGGTCA hexamer that is spaced by 10 bp. The pyrimidines in the -2 position relative to the core hexamer are both essential to function. In vitro binding studies of TRE1 showed no heterodimer formation with retinoid X receptor (RXR) beta or JEG nuclear extracts (containing RXR alpha) and bacterially expressed chicken T3 receptor alpha 1 (TR alpha) can occupy both half-sites although the 3' half-site is dominant. T3 causes dissociation of TR alpha from the 5' half-site but increases binding to the 3' half-site. Binding of a second TR to TRE1 is minimally cooperative; however, no cooperativity was noted for a functional mutant in which the half-sites are separated by 15 bp, implying that TRs bind as independent monomers. Nonetheless, T3 still causes TR dissociation from the DR+15, indicating that dissociation occurs independently of TR-TR contact and that rebinding of a T3-TR complex to the 3' half-site occurs because of its slightly higher affinity. A distal TRE (TRE2) is found at bp -700 and is a direct repeat of a PuGGTCA hexamer spaced by 4 bp. It has typical TR homodimer and TR-RXR heterodimer binding properties. The TRE1 of hdio1 is the first example of a naturally occurring TRE consisting of two relatively independent octamer sequences which do not require the RXR family of proteins for function.     

New sequential-assignment routes of nucleic acid NMR signals using a [5'-(13)C]-labeled DNA dodecamer

NMR signal assignments for DNA oligomers have been performed by the well-established sequential assignment procedures based on NOESY and COSY. The H4'/H5'/H5' resonance region is congested and difficult to analyze without the use of isotope-labeled DNA oligomers. Here a DNA dodecamer constructed with 2'-deoxy[5'-(13)C]ribonucleotides, 5'-d(*C*G*C*G*A*A*T*T*C*G*CG)-3' (*N = [5'-(13)C]Nucleotide), was prepared in an effort to analyze the H4'/H5'/H5' resonance region by 2D 1H-13C HMQC-NOESY. In the C5' and H1' resonance region, weak and strong cross peaks for C5'(i)-H1'(i) and C5'(i)-H1'(i-1), respectively, were found, thus enabling the sequential assignment within this region. A similar sequential assignment route was found between C5' and H2'. Proton pair distances evaluated from the canonical B-DNA as well as A-DNA indicated that these sequential-assignment routes on a 2D 1H-13C HMQC-NOESY spectrum work for most nucleic acid stem regions.     

Design of camp–CRP-activated promoters in Escherichia coli

We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by cAMP-CRP. Based on these observations, we propose that cAMP-CRP-activated promoters can be created by correctly aligning a CRP target and a -10 hexamer. This idea has been successfully tested by converting both a CRP-independent promoter and a sequence resembling the consensus -10 hexamer to strongly cAMP-CRP-activated promoters.     

The molecular chaperone, ClpA, has a single high affinity peptide binding site per hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.     

Interplay between AAUAAA and the trans-splice site in processing of a Caenorhabditis elegans operon pre-mRNA

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.     

Joining of 3'-modified oligonucleotides by T4 RNA ligase. Synthesis of a heptadecanucleotide corresponding to the bases 61--77 from Escherichia coli tRNAfMet.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.     

Uncharged stereoregular nucleic acid analogs: 2. Morpholino nucleoside oligomers with carbamate internucleoside linkages.

A novel oligonucleotide analog has been prepared from ribonucleoside derived morpholine subunits linked by carbamate groups. Oxidative cleavage of the 2',3' vicinal diol of cytidine followed by reductive amination of the resulting dialdehyde afforded the morpholine subunit. Coupling of the subunits are through carbamate moieties and the oligomers were characterized by 1H NMR and FAB MS. Evidence for interaction of the hexamer 19 with p(dG6) was found, but an atypical interaction of 19 with a RNA target was observed.     

Synthesis of nucleic acid methylphosphonates via the 1-hydroxybenzotriazole phosphotriester approach.

Dinucleoside methylphosphonates can easily be prepared starting from properly protected d-nucleosides and the bifunctional phosphorylating reagent methyl-O,O-bis(1-benzotriazolyl)phosphate. Separation of the diastereoisomers of 5'-DMTR-d-Ap(Me)T-3'-lev affords optically pure dinucleoside methylphosphonates which, after removal of the 3'-levulinoyl group, have been used for the synthesis of the two optically pure diastereoisomers of the hexamer d-CpGpAp(Me)TpCpG. Further, a one-pot procedure for the preparation of uridine-3',5'-cyclic methylphosphonate will be described. We also found that 3',5'-methylphosphonate linkages in RNA are not stable towards mild acid treatment.     

NMR studies on the binding of antitumor drug nogalamycin to DNA hexamer d(CGTACG).

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.     

Sequences at the 3' untranslated region of bamboo mosaic potexvirus RNA interact with the viral RNA-dependent RNA polymerase

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555-565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain (Delta 893) derived from BaMV open reading frame 1 could specifically bind to the 3' UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3' UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3' UTR showed that Delta 893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Delta 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.     

Complexation of single strand telomere and telomerase RNA template polyanions by deoxyribonucleic guanidine (DNG) polycations: plausible anticancer agents

Cancerous cell immortality is due to relatively high concentrations of telomerase enzyme which maintains telomere sequence during cell division. Deoxyribonucleic guanidine (DNG) is a positively charged DNA analog in which guanidine replaces the phosphordiester linkage of DNA. Mixed sequences of DNG and DNA oligonucleotides are referred to as chimera. Complexation of DNG and chimeric polycations with the complementary negatively charged non-coding telomere single strand d(5'-TTAGGG-3')(n) and the 11-base telomeric RNA template (5'-CUAACCCUAAC-3') in the active site of telomerase has been studied. Calculated by ensemble sampling simulations in GBMV solvent model, we found that binding of complementary DNG hexamer with telomere is favored over that of DNA-telomere by approximately 10(6)-fold and binding of chimera hexamer is favored by approximately 10(4)-fold. Binding of complementary DNG with telomeric RNA is favored by 43 kcal/mol over telomere substrate binding with telomeric RNA.     

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.     

Uridine recognition motifs of human equilibrative nucleoside transporters 1 and 2 produced in Saccharomyces cerevisiae

The sugar moiety of nucleosides has been shown to play a major role in permeant-transporter interaction with human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2). To better understand the structural requirements for interactions with hENT1 and hENT2, a series of uridine analogs with sugar modifications were subjected to an assay that tested their abilities to inhibit [3H]uridine transport mediated by recombinant hENT1 and hENT2 produced in Saccharomyces cerevisiae. hENT1 displayed higher affinity for uridine than hENT2. Both transporters barely tolerated modifications or inversion of configuration at C(3'). The C(2')-OH at uridine was a structural determinant for uridine-hENT1, but not for uridine-hENT2, interactions. Both transporters were sensitive to modifications at C(5') and hENT2 displayed more tolerance to removal of C(5')-OH than hENT1; addition of an O-methyl group at C(5') greatly reduced interaction with either hENT1 or hENT2. The changes in binding energies between transporter proteins and the different uridine analogs suggested that hENT1 formed strong interactions with C(3')-OH and moderate interactions with C(2')-OH and C(5')-OH of uridine, whereas hENT2 formed strong interactions with C(3')-OH, weak interactions with C(5')-OH, and no interaction with C(2')-OH.