Single Guanosine Research Articles

SiRNA-Mimetic Ratiometric pH (sMiRpH) Probes for Improving Cell Delivery and mRNA Knockdown.

Second-generation siRNA-mimetic ratiometric pH probes (sMiRpH-2) were developed by hybridizing a 3'-FAM-labeled 2'-OMe RNA strand with a 3'-Cy5-labeled 25mer RNA strand. These duplexes demonstrated the silencing of cytoplasmic mRNA targets in HeLa cells as measured by RT-qPCR and supported by western blot analysis. Fluorescence intensity and lifetime measurements revealed that a single guanosine (G) positioned adjacent to FAM achieves substantial static quenching at pH 5, with additional collisional quenching rendering the dye almost nonemissive. A FAM-G π-π stacking interaction was evidenced by a red-shifted absorbance spectrum for FAM. Decreased quenching at near-neutral pH enhances the FAM dynamic range in the physiologic pH window and improves the differentiation in cells between endocytic entrapment and cytoplasmic release. Flow cytometric analysis of intracellular pH and uptake using sMiRpH-2 was corroborated by live cell confocal microscopy and found to be predictive of knockdown efficacy. A sMiRpH-2 probe successfully predicted the relative efficacy of two transfection agents in more challenging SK-OV-3 cells, which highlights its use for the rapid assessment of nonviral siRNA delivery vectors.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Deadenylation kinetics of mixed poly(A) tails at single-nucleotide resolution

Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or ‘mixed tailing’, has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4–NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4–NOT stalls at the 0, −1 and −2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.

Fidelity of base-pair recognition by a 3'-5' polymerase: mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase.

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3′–5′ addition of a single nontemplated guanosine (G−1) to the 5′ end of tRNAHis. In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3′–5′ polymerization of Watson–Crick (WC) base pairs, utilizing nucleotides in the 3′-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3′–5′ polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3′–5′ polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA “template,” addition of a non-WC G−1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5′–3′ polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3′–5′ polymerases.

Structural basis for transcriptional start site control of HIV-1 RNA fate.

Heterogeneous transcriptional start site usage by HIV-1 produces 5'-capped RNAs beginning with one, two, or three 5'-guanosines (Cap1G, Cap2G, or Cap3G, respectively) that are either selected for packaging as genomes (Cap1G) or retained in cells as translatable messenger RNAs (mRNAs) (Cap2G and Cap3G). To understand how 5'-guanosine number influences fate, we probed the structures of capped HIV-1 leader RNAs by deuterium-edited nuclear magnetic resonance. The Cap1G transcript adopts a dimeric multihairpin structure that sequesters the cap, inhibits interactions with eukaryotic translation initiation factor 4E, and resists decapping. The Cap2G and Cap3G transcripts adopt an alternate structure with an elongated central helix, exposed splice donor residues, and an accessible cap. Extensive remodeling, achieved at the energetic cost of a G-C base pair, explains how a single 5'-guanosine modifies the function of a ~9-kilobase HIV-1 transcript.

One guanosine determines transcript fate

RNA Structure Transcripts of the HIV-1 RNA genome can be either spliced and translated into viral proteins or packaged into new virions as a progeny genome. The path taken depends on whether the transcript contains one guanosine at the 5′ terminus (1G) rather than two or three (2G or 3G). Brown et al. used nuclear magnetic resonance spectroscopy to show that 1G transcripts adopt a dimeric structure that sequesters a terminal cap required for translation and splicing but exposes sites that bind to the HIV-1 Gag protein, which recruits the genome during viral assembly. Conversely, 2G or 3G transcripts have the cap accessible, but Gag-binding sites are sequestered. Therefore, a single guanosine acts as a conformational switch to determine the fate of HIV-1 transcripts. Science , this issue p. [413][1] [1]: /lookup/doi/10.1126/science.aaz7959

METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation.

Summary7-methylguanosine (m7G) is present at mRNA caps and at defined internal positions within tRNAs and rRNAs. However, its detection within low-abundance mRNAs and microRNAs (miRNAs) has been hampered by a lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in miRNAs. Using this technique (Borohydride Reduction sequencing [BoRed-seq]) alongside RNA immunoprecipitation, we identify m7G within a subset of miRNAs that inhibit cell migration. We show that the METTL1 methyltransferase mediates m7G methylation within miRNAs and that this enzyme regulates cell migration via its catalytic activity. Using refined mass spectrometry methods, we map m7G to a single guanosine within the let-7e-5p miRNA. We show that METTL1-mediated methylation augments let-7 miRNA processing by disrupting an inhibitory secondary structure within the primary miRNA transcript (pri-miRNA). These results identify METTL1-dependent N7-methylation of guanosine as a new RNA modification pathway that regulates miRNA structure, biogenesis, and cell migration.

English

Beta-thalassemia major (β-TM) in children is associated with an increase in the risk of premature cardiovascular complications caused by accelerated atherosclerosis which significantly contributes to morbidity and mortality. The molecular mechanisms underlying β-TM associated atherosclerosis and its relation to biochemical risk factors still remain obscure. We aimed to investigate the association between SIRT1 single nucleotide polymorphism (SNP), lipid profile, ferritin, malondialdehyde (MDA), 8-hydroxydeoxy guanosine (8-OHdG) and total antioxidant capacity (TAC) in relation to carotid intima media thickness (CIMT) trying to explain some mechanisms of cardiovascular complication in beta-Thalassemia major (β-TM) children. This study was carried out on 100 Egyptian children with β-TM (Group II) subdivided equally according to CIMT into Group IIa, with CIMT<0.5 mm and Group IIb with CIMT ≥ 0.5 mm, in addition to 50 healthy children as controls (Group I). All groups were subjected to measurement of plasma Ferritin, lipid profile, MDA and TAC colorimetrically. 8-OHdG levels were estimated by enzyme-linked immunosorbent assay (ELISA) in addition genotyping pattern for the SIRT1 (rs7069102) SNP was evaluated using PCR-CTPP technique. Values for AIP, ferritin, MDA and 8-OHdG levels, were significantly higher in β-TM groups compared to control with higher levels in patients with CIMT more than 0.5 mm but values of TAC and (HDL-C) showed significant decrease. CIMT was significantly correlated with age, atherogenic index of plasma (AIP), ferritin, MDA and 8-OHdG, HDL-C and TAC. There was significant difference in C allele distribution of SIRT1 rs7096102 was 57% in control subjects, 29% in Group IIa and 23% in Group IIb. However, the allele G distribution was 43% in control subjects, 71% in Group IIa and 77% Group IIb. Significant association of SIRT1 (rs7069102) polymorphism with dyslipidemia, ferritin and oxidative stress may conduct atherosclerotic potential in β-TM by affecting the severity of CIMT. Key words: Beta-thalassemia major, carotid intima media thickness.

Morphological diversity of supramolecular polymers of DNA-containing oligopyrenes - formation of chiroptically active nanosheets.

The effect of DNA on the morphology of supramolecular polymers assembled from chimeric DNA-pyrene oligophosphodiesters in water is described. The number of deoxynucleotides determines if the self-assembly yields 1D or 2D structures. A single guanosine nucleotide attached to a heptapyrenotide induces chiroptical activity in self-assembled nanosheets.

Saccharomyces cerevisiae Thg1 Uses 5′-Pyrophosphate Removal To Control Addition of Nucleotides to tRNAHis

In eukaryotes, the tRNAHis guanylyltransferase (Thg1) catalyzes 3′–5′ addition of a single guanosine residue to the −1 position (G–1) of tRNAHis, across from a highly conserved adenosine at position 73 (A73). After addition of G–1, Thg1 removes pyrophosphate from the tRNA 5′-end, generating 5′-monophosphorylated G–1-containing tRNA. The presence of the 5′-monophosphorylated G–1 residue is important for recognition of tRNAHis by its cognate histidyl-tRNA synthetase. In addition to the single-G–1 addition reaction, Thg1 polymerizes multiple G residues to the 5′-end of tRNAHis variants. For 3′–5′ polymerization, Thg1 uses the 3′-end of the tRNAHis acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3′-OH from each incoming NTP on the intact 5′-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5′-pyrophosphate removal and 3′–5′ polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNAHis substrates in which the identity of the N–1:N73 base pair was varied to mimic various products of the N–1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5′-triphosphate is correlated with efficient 3′–5′ reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the −1 position with wild-type tRNAHis is proposed.

Electron-Hole Transfer in G-Quadruplexes with Different Tetrad Stacking Geometries: A Combined QM and MD Study

G-quadruplex nucleic acids represent a unique avenue for the building of electrically conductive wires. These four-stranded structures are formed through the stacking of multiple planar guanine assemblies termed G-tetrads. The diverse folding patterns of G-quadruplexes allow for several geometries to be adopted by stacked guanine bases within the core and at the dimeric interface of these structures. It is currently not clear how different G-tetrad stacking arrangements affect electron hole mobility through a G-quadruplex wire. Using a combined quantum mechanics and molecular dynamics approach, we demonstrate that the electron-hole transfer rates within the G-tetrad stacks vary greatly for different stacking geometries. We identify a distinguished structure that allows for strong electronic coupling and thus enhanced molecular electric conductance. We also demonstrate the importance of sampling a large number of geometries when considering the bulk properties of such systems. Hole hopping within single G-tetrads is slower by at least two orders of magnitude than between stacked guanines; therefore, hole jumping within individual tetrads should not affect the hole mobility in G-quadruplexes. The results of this study suggest engineering G-tetrads with continuous 5/6-ring stacking from an assembly of single guanosine analogs or through modification of the backbone in G-rich DNA sequences.

A thyroid peroxidase (TPO) mutation in dogs reveals a canid-specific gene structure

Congenital hypothyroidism with goiter (CHG) occurring as an autosomal recessive disorder is typically due to a defect of thyroid hormone synthesis (aka dyshormonogenesis). Thyroid peroxidase (TPO) is a multifunctional, heme-containing enzyme whose activity is required, and several inactivating TPO mutations causing CHG in humans and dogs have been described. Recently, two half-sib Spanish water dog (SWD) pups were diagnosed with CHG based on clinical signs, endocrine testing, and thyroid histology. TPO enzyme activity was absent, and immuno-cross-reactive TPO was undetectable in affected-dog thyroid tissue. A single guanosine insertion was observed in the first exon of the affected-dog TPO cDNA at a site not previously thought to be within the coding sequence. The insertion allele segregated with the deduced disease allele in the SWD breed and was not observed in unrelated dogs of various breeds. Comparison of the insertion site (an 8-nt poly-G tract) with the orthologous sequences of other mammalian reference genomes revealed that the octa-G tract obliterated the intron 1 splice acceptor site and the exon 2 translation initiation codon found at that position in other species. An in-frame ATG in strong Kozak consensus context was observed in the normal dog sequence 12 codons 5' of the usual mammalian start site, suggesting that dogs have lost the noncoding exon 1 demonstrated in human and mouse. A survey of TPO sequences in other carnivore species indicates that the poly-G tract necessitating an alternative translation initiation site is a canid-specific feature.

The 2′-O-methylation status of a single guanosine controls transfer RNA–mediated Toll-like receptor 7 activation or inhibition

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2'-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus-infected PBMCs.

Kinetic Analysis of 3′–5′ Nucleotide Addition Catalyzed by Eukaryotic tRNAHis Guanylyltransferase

The tRNA(His) guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the -1 position (G(-1)) of tRNA(His), using an unusual 3'-5' nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3'-5' addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3'-5' nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G(-1) addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (k(aden)), nucleotidyl transfer (k(ntrans)), and removal of pyrophosphate from the G(-1)-containing tRNA (k(ppase)). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G(-1) addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNA(His) maturation.

Association of the plasminogen activator inhibitor-1(PAI-1) gene 4G/5G promoter polymorphism in Buerger's disease (Tromboangiitis obliterans)

Thromboangiitis obliterans (TAO) is an unusual tobacco-associated vasculopathy that is a non- atherosclerotic inflammatory disorder of unkn- own etiology that affects small and medium- sized vessels of the extremities. The single guanosine nucleotide deletion/insertion polym- orphism (4G/5G) at -675 bp in promoter region of the PAI-1 gene is the major genetic determi- nant of PAI-1 expression. Plasma PAI-1 level is higher in people with the homozygous 4G genotype than in those with the 5G/5G genotype and renders higher transcription activity. The aim of this study was to determine the status and the role of PAI-1 gene 4G/5G promoter polymorphism in patients with Buerger's disease (Thromboangiitis obliterans—TAO). The current case-control study included 30 consecutive pat- ients with Buerger's disease (mean age 42.9 ± 14.3 years, 28 men and 2 women), and 30 healthy volunteers (mean age 40.9 ± 4.79 years, 27 men and 3 women) between January 2006 and September 2009. Patients and control cases were genotyped for the 4G/5G polymorphism using the multiplex PCR based stripassay reverse hybridisation technique. It was found that heterozygote PAI-1 gene polymorphisms (p < 0.05) was significantly more frequent in patients with TAO in the current results. There was a significant difference in genotype distribution between the two groups (P < 0.001). The 4G allele occurred more frequently in the patient group of heavy smokers (P = 0.05). The current study shows the higher prevalence of of 4G allele in TAO patients in Sivas population means gene may predispose to TAO.

Plasminogen activator inhibitor‐1 and asthma: role in the pathogenesis and molecular regulation

Plasminogen activator inhibitor (PAI)-1 is a major inhibitor of the fibrinolytic system. PAI-1 levels are markedly increased in asthmatic airways, and mast cells (MCs), a pivotal cell type in the pathogenesis of asthma, are one of the main sources of PAI-1 production. Recent studies suggest that PAI-1 may promote the development of asthma by regulating airway remodelling, airway hyperresponsiveness (AHR), and allergic inflammation. The single guanosine nucleotide deletion/insertion polymorphism (4G/5G) at -675 bp of the PAI-1 gene is the major genetic determinant of PAI-1 expression. Plasma PAI-1 level is higher in people with the 4G/4G genotype than in those with the 5G/5G genotype. A strong association between the 4G/5G polymorphism and the risk and the severity of asthma has been suggested. Levels of plasma IgE and PAI-1 and severity of AHR are greater in asthmatic patients with the 4G/4G genotype than in those with the 5G/5G genotype. The PAI-1 promoter with the 4G allele renders higher transcription activity than the PAI-1 promoter with the 5G allele in stimulated MCs. The molecular mechanism for the 4G allele-mediated higher PAI-1 expression is associated with greater binding of upstream stimulatory factor-1 to the E-box adjacent to the 4G site (E-4G) than to the E-5G. In summary, PAI-1 may play an important role in the pathogenesis of asthma. Further studies evaluating the mechanisms of PAI-1 action and regulation may lead to the development of a novel prognostic factor and therapeutic target for the treatment and prevention of asthma and other PAI-1-associated diseases.

No association between the promoter polymorphisms of PAI-1 gene and sporadic Alzheimer's disease in Chinese Han population

Amyloid beta peptide (Aβ) accumulation is the major event in the brain of cases with Alzheimer's disease (AD). The serine protease plasmin, which is generated from the inactive zymogen plasminogen, could accelerate Aβ degradation, and thus may play a role in AD pathogenesis. It has been reported that the increasing of plasminogen activator inhibitor-1 (PAI-1) inhibits the activity of plasminogen activator and thus reduces the generation of plasmin in vivo. For seeking the correlation of the PAI-1 promoter with sporadic AD (SAD), we performed a case–control study in a group of Chinese Han population. In the study, we detected two polymorphisms, a G/A single base substitution polymorphism at −844 bp (rs2227631) and a single guanosine deletion/insertion 4G/5G polymorphism at −675 bp (rs1799889) upstream from the start of transcription. Direct sequencing was used for genotyping in 324 SAD patients and 278 controls. We failed to find any associations between these two polymorphisms and SAD even after stratified by age of onset, gender and apolipoprotein E (APOE) ɛ4 status. Our data do not support that there is an association between the PAI-1 promoter polymorphisms and SAD in the Chinese Han population.

The impact of the PAI-1 4G/5G polymorphism on the outcome of patients with ALI/ARDS

Intoduction Increased levels of plasminogen activator inhibitor-1 (PAI-1) have been associated with worse outcome in ALI/ARDS. A single guanosine insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene, may play an important role in the regulation of PAI-1 expression. The objective of the study was to evaluate the effect of this polymorphism on the outcome of critically ill patients with ALI/ARDS. Materials and Methods 52 consecutive ventilated patients with ALI/ARDS were studied. Bronchoalveolar lavage was performed within 48 hours from diagnosis. Measurement of plasma and BALF PAI-1 activity and D-dimers levels, and 4G/5G genotyping of PAI-1 were carried out. The primary outcome was 28-day mortality, and secondary outcomes included organ dysfunction and ventilator-free days. Results 17 patients were homozygotes for the 4G allele. Severity scores were not different between subgroups upon study enrollment. 28-day mortality was 70.6% and 42.9% for the 4G-4G and the non-4G-4G patients, respectively (p = 0.06). PAI-1 activity levels and D-dimer in plasma and BALF were not significantly different between the 4G-4G and the non-4G-4G subgroups. In the multivariate analysis, genotype 4G/4G was the only variable independently associated with 28-day mortality (Odds Ratio = 9.95, 95% CI: 1.79-55.28, p = 0.009). Furthermore, genotype 4G/4G and plasma PAI-1 activity levels were independently negatively associated with ventilator free days (p = 0.033 and p = 0.008, respectively). Conclusions ALI/ARDS patients, homozygous for the 4G allele of the PAI-1 gene, experienced higher 28-day mortality. This genotype was associated with a reduction in the number of days of unassisted ventilation and was inversely associated with the number of days without organ failure.

Conformationally restricted nucleotides as a probe of structure–function relationships in RNA

We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed. We apply LNA probing at multiple sites to analyze self-cleavage in the lead-dependent ribozyme (leadzyme), thermodynamic stability in the UUCG tetraloop, and the kinetics of recognition of U1A protein by U1 snRNA hairpin II. In the leadzyme, locking a single guanosine residue into the C3'-endo pucker increases the catalytic rate by a factor of 20, despite the fact that X-ray crystallographic and NMR structures of the leadzyme ground state reported a C2'-endo conformation at this site. These results strongly suggest that a conformational change at this position is critical for catalytic function. Functional insights obtained in all three systems demonstrate the highly general applicability of LNA probing in analysis of the role of ribose orientation in RNA structure, dynamics, and function.

Cis -Diammine(pyridine)chloroplatinum(II), a monofunctional platinum(II) antitumor agent: Uptake, structure, function, and prospects

We have identified unique chemical and biological properties of a cationic monofunctional platinum(II) complex, cis-diammine(pyridine)chloroplatinum(II), cis-[Pt(NH(3))(2)(py)Cl](+) or cDPCP, a coordination compound previously identified to have significant anticancer activity in a mouse tumor model. This compound is an excellent substrate for organic cation transporters 1 and 2, also designated SLC22A1 and SLC22A2, respectively. These transporters are abundantly expressed in human colorectal cancers, where they mediate uptake of oxaliplatin, cis-[Pt(DACH)(oxalate)] (DACH = trans-R,R-1,2-diaminocyclohexane), an FDA-approved first-line therapy for colorectal cancer. Unlike oxaliplatin, however, cDPCP binds DNA monofunctionally, as revealed by an x-ray crystal structure of cis-{Pt(NH(3))(2)(py)}(2+) bound to the N7 atom of a single guanosine residue in a DNA dodecamer duplex. Although the quaternary structure resembles that of B-form DNA, there is a base-pair step to the 5' side of the Pt adduct with abnormally large shift and slide values, features characteristic of cisplatin intrastrand cross-links. cDPCP effectively blocks transcription from DNA templates carrying adducts of the complex, unlike DNA lesions of other monofunctional platinum(II) compounds like {Pt(dien)}(2+). cDPCP-DNA adducts are removed by the nucleotide excision repair apparatus, albeit much less efficiently than bifunctional platinum-DNA intrastrand cross-links. These exceptional characteristics indicate that cDPCP and related complexes merit consideration as therapeutic options for treating colorectal and other cancers bearing appropriate cation transporters.