Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH

Identification of multiple genomic DNA sequences which form i-motif structures at neutral pH

i-Motifs are alternative DNA secondary structures formed in cytosine-rich sequences. Particular examples of these structures, traditionally assumed to be stable only at acidic pH, have been found to form under near-physiological conditions. To determine the potential impact of these structures on physiological processes, investigation of sequences with the capacity to fold under physiological conditions is required. Here we describe a systematic study of cytosine-rich DNA sequences, with varying numbers of consecutive cytosines, to gain insights into i-motif DNA sequence and structure stability. i-Motif formation was assessed using ultraviolet spectroscopy, circular dichroism and native gel electrophoresis. We found that increasing cytosine tract lengths resulted in increased thermal stability; sequences with at least five cytosines per tract folded into i-motif at room temperature and neutral pH. Using these results, we postulated a folding rule for i-motif formation, analogous to (but different from) that for G-quadruplexes. This indicated that thousands of cytosine-rich sequences in the human genome may fold into i-motif structures under physiological conditions. Many of these were found in locations where structure formation is likely to influence gene expression. Characterization of a selection of these identified i-motif forming sequences uncovered 17 genomic i-motif forming sequence examples which were stable at neutral pH

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming region

Direct visualization of G-quadruplexes in DNA using atomic force microscopy

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sγ3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation

Reliability of Therapist Effects in Practice-Based Psychotherapy Research : A Guide for the Planning of Future Studies

This paper aims to provide researchers with practical information on sample sizes for accurate estimations of therapist effects (TEs). The investigations are based on an integrated sample of 48,648 patients treated by 1800 therapists. Multilevel modeling and resampling were used to realize varying sample size conditions to generate empirical estimates of TEs. Sample size tables, including varying sample size conditions, were constructed and study examples given. This study gives an insight into the potential size of the TE and provides researchers with a practical guide to aid the planning of future studies in this field

Left: frequency distributions of loops of lengths 1–7 bases for the entire human genome

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of quadruplexes in the human genome"</p><p>Nucleic Acids Research 2005;33(9):2908-2916.</p><p>Published online 24 May 2005</p><p>PMCID:PMC1140081.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Right: percentage excesses of loop 2 counts over the averages of loops 1 and 3 for the entire human genome

Ethical principles and a practical approach to support policy making through the next phases of the COVID-19 pandemic and beyond.

There is an urgent need for an ethical framework to help us address the local and national challenges that we face as clinicians during the COVID-19 pandemic. We propose four key commitments from which a practical and consistent ethical approach can be derived. These commitments are to articulate the needs, rights and interests of the different stakeholders affected by any policy; to be accountable and transparent, recognising that people are autonomous individuals with values and concerns of their own; to consider the impact of our actions on the sustainability of the NHS, infrastructure, service demands and staff welfare; and to treat everybody equitably, with all deserving of consideration and care. Implementing these commitments will require a number of specific actions. We must put in place frameworks enabling clear advocacy for each competing objective; communicate policy and practice effectively to the public; promote integration of decision-making among social, primary, secondary and tertiary care and reduce or stop unnecessary or inefficient interventions; minimise health inequalities; and build spare capacity into the system.In this article, we expand on these actions, and note the legal context in which this would be delivered

Distance-dependent duplex DNA destabilization proximal to G-quadruplex/i-motif sequences

G-quadruplexes and i-motifs are complementary examples of non-canonical nucleic acid substructure conformations. G-quadruplex thermodynamic stability has been extensively studied for a variety of base sequences, but the degree of duplex destabilization that adjacent quadruplex structure formation can cause has yet to be fully addressed. Stable in vivo formation of these alternative nucleic acid structures is likely to be highly dependent on whether sufficient spacing exists between neighbouring duplex- and quadruplex-/i-motif-forming regions to accommodate quadruplexes or i-motifs without disrupting duplex stability. Prediction of putative G-quadruplex-forming regions is likely to be assisted by further understanding of what distance (number of base pairs) is required for duplexes to remain stable as quadruplexes or i-motifs form. Using oligonucleotide constructs derived from precedented G-quadruplexes and i-motif-forming bcl-2 P1 promoter region, initial biophysical stability studies indicate that the formation of G-quadruplex and i-motif conformations do destabilize proximal duplex regions. The undermining effect that quadruplex formation can have on duplex stability is mitigated with increased distance from the duplex region: a spacing of five base pairs or more is sufficient to maintain duplex stability proximal to predicted quadruplex/i-motif-forming regions