Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates.

Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis.

Most RNA folding studies have been performed under non-physiological conditions of high concentrations (≥10 mM) of Mg2+free, while actual cellular concentrations of Mg2+free are only ~1 mM in a background of greater than 50 mM Mg2+total. To uncover cellular behavior of RNA, we devised cytoplasm mimic systems that include biological concentrations of amino acids, which weakly chelate Mg2+. Amino acid-chelated Mg2+ (aaCM) of ~15 mM dramatically increases RNA folding and prevents RNA degradation. Furthermore, aaCM enhance self-cleavage of several different ribozymes, up to 100,000-fold at Mg2+free of just 0.5 mM, indirectly through RNA compaction. Other metabolites that weakly chelate magnesium offer similar beneficial effects, which implies chelated magnesium may enhance RNA function in the cell in the same way. Overall, these results indicate that the states of Mg2+ should not be limited to free and bound only, as weakly bound Mg2+ strongly promotes RNA function under cellular conditions.

Complexity in pH-Dependent Ribozyme Kinetics: Dark pKa Shifts and Wavy Rate-pH Profiles.

Charged bases occur in RNA enzymes, or ribozymes, where they play key roles in catalysis. Cationic bases donate protons and perform electrostatic catalysis, while anionic bases accept protons. We previously published simulations of rate-pH profiles for ribozymes in terms of species plots for the general acid and general base that have been useful for understanding how ribozymes respond to pH. In that study, we did not consider interaction between the general acid and general base or interaction with other species on the RNA. Since that report, diverse small ribozyme classes have been discovered, many of which have charged nucleobases or metal ions in the active site that can either directly interact and participate in catalysis or indirectly interact as "influencers". Herein, we simulate experimental rate-pH profiles in terms of species plots in which reverse protonated charged nucleobases interact. These analyses uncover two surprising features of pH-dependent enzyme kinetics. (1) Cooperativity between the general acid and general base enhances population of the functional forms of a ribozyme and manifests itself as hidden or "dark" pKa shifts, real pKa shifts that accelerate the reaction but are not readily observed by standard experimental approaches, and (2) influencers favorably shift the pKas of proton-transferring nucleobases and manifest themselves as "wavy" rate-pH profiles. We identify parallels with the protein enzyme literature, including reverse protonation and wavelike behavior, while pointing out that RNA is more prone to reverse protonation. The complexities uncovered, which arise from simple pairwise interactions, should aid deconvolution of complex rate-pH profiles for RNA and protein enzymes and suggest veiled catalytic devices for promoting catalysis that can be tested by experiment and calculation.

Cooperative Interactions in the Hammerhead Ribozyme Drive pKa Shifting of G12 and Its Stacked Base C17.

General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2'-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2'-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5'-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.

Eliminating blurry bands in gels with a simple cost-effective repair to the gel cassette.

Gel electrophoresis and subsequent imaging using phosphorimagers is one of the most important and widely used techniques in RNA and DNA analysis. Radiolabeling nucleic acids with 32P and detecting bands using a phoshorimager are useful both in a qualitative sense for nucleic acid detection and in a quantitative sense for structural, kinetic, or binding-based assays. Because of this, good resolution of gel bands based on molecular weight and size of RNA or DNA is essential for analysis. The appearance of blurry gel bands of 32P-labeled RNA and DNA thus represents a serious problem in the laboratory. A quick search on the Internet uncovers numerous reports begrudging the appearance of blurry bands, as well as attempts to fix them without success. Indeed, our laboratories were beset by the intermittent problem of blurry gels for over one year before we found a solution. Herein we describe a simple and cost-effective solution to a problem that we show originates from the phosphorimager cassettes rather than the integrity of the gel itself. We hope that the information provided here will lead to immediate help for other laboratories experiencing similar issues with labeled nucleic acid gel-based assays. The improvement in the clarity of the gels is nothing short of astonishing in many instances and will lead to higher resolution images for analysis and publications.

Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²âº, Nucleotides, and RNA.

Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned ∼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments.

Potential of pretreatment neural activity in the visual cortex during emotional processing to predict treatment response to scopolamine in major depressive disorder.

CONTEXT: The need for improved treatment options for patients with major depressive disorder (MDD) is critical. Faster-acting antidepressants and biomarkers that predict clinical response will facilitate treatment. Scopolamine produces rapid antidepressant effects and thus offers the opportunity to characterize potential biomarkers of treatment response within short periods. OBJECTIVE: To determine if baseline brain activity when processing emotional information can predict treatment response to scopolamine in MDD. DESIGN: A double-blind, placebo-controlled, crossover study together with repeated functional magnetic resonance imaging, acquired as participants performed face-identity and face-emotion working memory tasks. SETTING: National Institute of Mental Health Division of Intramural Research Programs. PARTICIPANTS: Fifteen currently depressed outpatients meeting DSM-IV criteria for recurrent MDD and 21 healthy participants, between 18 and 55 years of age. MAIN OUTCOME MEASURE: The magnitude of treatment response to scopolamine (percentage of change in the Montgomery-Asberg Depression Rating Scale score between study end and baseline) was correlated with blood oxygen level-dependent (BOLD) signal associated with each working memory component (encode, maintenance, and test) for both identity and emotion tasks. Treatment response also was correlated with change in BOLD response (scopolamine vs baseline). Baseline activity was compared between healthy and MDD groups. RESULTS: Baseline BOLD response in the bilateral middle occipital cortex, selectively during the stimulus-processing components of the emotion working memory task (no correlation during the identity task), correlated with treatment response magnitude. Change in BOLD response following scopolamine administration in overlapping areas in the middle occipital cortex while performing the same task conditions also correlated with clinical response. Healthy controls showed higher activity in the same visual regions than patients with MDD during baseline. CONCLUSION: These results implicate cholinergic and visual processing dysfunction in the pathophysiology of MDD and suggest that neural response in the visual cortex, selectively to emotional stimuli, may provide a useful biomarker for identifying patients who will respond favorably to scopolamine. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00055575.

Baseline mood-state measures as predictors of antidepressant response to scopolamine.

Identifying predictors of antidepressant response will facilitate the successful treatment of patients suffering from depression. Scopolamine produces robust antidepressant responses in unipolar and bipolar depression. Here we evaluate the potential for baseline self-ratings to predict treatment response to scopolamine. Fifty-one unipolar and bipolar patients participated in a double-blind, placebo-controlled crossover trial. Following a single-blind placebo session, participants randomly received P/S or S/P (P=3 placebo; S=3 scopolamine (4µg/kg) sessions). Mood-state self-ratings (Profile of Mood State (POMS) and Visual Analog Scales (VAS)) and depression severity (Montgomery-Åsberg Depression Rating Scale (MADRS)) were obtained before each infusion. Day 1 (baseline/placebo) self-ratings were used in a discriminant function analysis to identify linear combinations of individual items that predict response. The discriminant analysis significantly separated responders from non-responders in both the unipolar and bipolar diagnostic subgroups. The discriminant functions accurately classified over 85% of patients as responders/non-responders. The POMS depression subscale significantly correlated with clinical response, as did the VAS restlessness, sad, and irritated scales. These results indicate that self-report mood-ratings obtained before treatment can predict response outcome to scopolamine, and suggest that a constellation of mood-state features may be related to clinical response.

Course of subthreshold bipolar disorder in youth: diagnostic progression from bipolar disorder not otherwise specified.

OBJECTIVE: To determine the rate of diagnostic conversion from an operationalized diagnosis of bipolar disorder not otherwise specified (BP-NOS) to bipolar I disorder (BP-I) or bipolar II disorder (BP-II) in youth over prospective follow-up and to identify factors associated with conversion. METHOD: Subjects were 140 children and adolescents recruited from clinical referrals or advertisement who met operationalized criteria for BP-NOS at intake and participated in at least one follow-up evaluation (91% of initial cohort). Diagnoses were assessed at follow-up interviews using the Longitudinal Interval Follow-Up Evaluation. The mean duration of follow-up was 5 years and the mean interval between assessments was 8.2 months. RESULTS: Diagnostic conversion to BP-I or BP-II occurred in 63 subjects (45%): 32 (23%) to BP-I (nine of whom had initially converted to BP-II) and 31 to only BP-II (22%). Median time from intake to conversion was 58 weeks. First- or second-degree family history of mania or hypomania was the strongest baseline predictor of diagnostic conversion (p = .006). Over follow-up, conversion was associated with greater intensity of hypomanic symptoms and with greater exposure to specialized, intensive outpatient psychosocial treatments. There was no association between conversion and exposure to treatment with particular medication classes. CONCLUSIONS: Children and adolescents referred with mood symptoms that meet operationalized criteria for BP-NOS, particularly those with a family history of BP, frequently progress to BP-I or BP-II. Efforts to identify these youth and effectively intervene may have the potential to curtail the progression of mood disorders in this high-risk population.