Mono- and bilayer smectic liquid crystal ordering in dense solutions of "gapped" DNA duplexes.

Although its mesomorphic properties have been studied for many years, only recently has the molecule of life begun to reveal the true range of its rich liquid crystalline behavior. End-to-end interactions between concentrated, ultrashort DNA duplexes-driving the self-assembly of aggregates that organize into liquid crystal phases-and the incorporation of flexible single-stranded "gaps" in otherwise fully paired duplexes-producing clear evidence of an elementary lamellar (smectic-A) phase in DNA solutions-are two exciting developments that have opened avenues for discovery. Here, we report on a wider investigation of the nature and temperature dependence of smectic ordering in concentrated solutions of various "gapped" DNA (GDNA) constructs. We examine symmetric GDNA constructs consisting of two 48-base pair duplex segments bridged by a single-stranded sequence of 2 to 20 thymine bases. Two distinct smectic layer structures are observed for DNA concentration in the range [Formula: see text] mg/mL. One exhibits an interlayer periodicity comparable with two-duplex lengths ("bilayer" structure), and the other has a period similar to a single-duplex length ("monolayer" structure). The bilayer structure is observed for gap length ≳10 bases and melts into the cholesteric phase at a temperature between 30 °C and 35 °C. The monolayer structure predominates for gap length ≲10 bases and persists to [Formula: see text]C. We discuss models for the two layer structures and mechanisms for their stability. We also report results for asymmetric gapped constructs and for constructs with terminal overhangs, which further support the model layer structures.

Rationally designed DNA therapeutics can modulate human TH expression by controlling specific GQ formation in its promoter.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the catecholamine (CA) biosynthesis pathway, making TH a molecular target for controlling CA production, specifically dopamine. Dysregulation of dopamine is correlated with neurological diseases such as Parkinson's disease (PD) and post-traumatic stress disorder (PTSD), among others. Previously, we showed that a 49-nucleotide guanine (G)-rich sequence within the human TH promoter adopts two different sets of G-quadruplex (GQ) structures (5'GQ and 3'GQ), where the 5'GQ uses G-stretches I, II, IV, and VI in TH49, which enhances TH transcription, while the 3'GQ utilizes G-stretches II, IV, VI, and VII, which represses transcription. Herein, we demonstrated targeted switching of these GQs to their active state using rationally designed DNA GQ Clips (5'GQ and 3'GQ Clips) to modulate endogenous TH gene expression and dopamine production. As a translational approach, we synthesized a targeted nanoparticle delivery system to effectively deliver the 5'GQ Clip in vivo. We believe this strategy could potentially be an improved approach for controlling dopamine production in a multitude of neurological disorders, including PD.

A piRNA utilizes HILI and HIWI2 mediated pathway to down-regulate ferritin heavy chain 1 mRNA in human somatic cells.

The piwi interacting RNAs (piRNAs) are small non-coding RNAs that specifically bind to the PIWI proteins, a functional requirement. The piRNAs regulate germline development, transposons control, and gene expression. However, piRNA-mediated post-transcriptional gene regulation in human somatic cells is not well understood. We discovered a human piRNA (piR-FTH1) which has a complementary sequence in the ferritin heavy chain 1 (Fth1) mRNA. We demonstrated that expression of piR-FTH1 and Fth1 are inversely correlated in the tested tumor cell lines. We found that piR-FTH1 negatively regulates the Fth1 expression at post-transcriptional level in triple negative breast cancer (TNBC) cells. Additionally, we confirmed that transfected piR-FTH1 knocks down the Fth1 mRNA via the HIWI2 and HILI mediated mechanism. piR-FTH1 mediated Fth1 repression also increased doxorubicin sensitivity by a remarkable 20-fold in TNBC cells. Since the current piRNA-mediated knockdowns of target mRNA are mostly reported in germ line cells, piRNA-mediated post-transcriptional gene regulation in somatic cells is rather unique in its application and mechanistically uses an alternative pathway to siRNA and miRNA. This work begins to lay the groundwork with a broader impact on treatment of various diseases that are linked to elevated levels of specific mRNAs which have a piRNA target.

An Independently folding RNA G-quadruplex domain directly recruits the 40S ribosomal subunit.

In this study, we report that a 17-nucleotide independently folding RNA G-quadruplex (GQ) domain within the 294-nucleotide human VEGF IRES A interacts with the 40S ribosomal subunit. Footprinting and structure mapping analyses indicate that the RNA GQ forms independently and interacts directly with the 40S ribosomal subunit in the absence of other protein factors. Moreover, a filter binding assay in conjunction with enzymatic footprinting clearly established that the GQ-forming domain singularly dictates the binding affinity and also the function of internal ribosomal entry site (IRES) A. The deletion of the GQ domain abrogates the binding of the 40S ribosomal subunit to the IRES, which impairs cap-independent translation initiation. The findings provide a unique and defined role for a noncanonical RNA structure in cap-independent translation initiation by cellular IRESs. The GQ structure when present in an IRES acts as an essential element in contrast to their generally accepted inhibitory role in translation. The results of this study explain the hitherto unknown mechanistic necessity of the GQ structure in IRES function.

G-Quadruplex-Enabling Sequence within the Human Tyrosine Hydroxylase Promoter Differentially Regulates Transcription.

G-Quadruplexes (GQs) found within the promoter regions of genes are known to mostly act as repressors of transcription. Here we report a guanosine (G)-rich segment in the 3'-proximal promoter region of human tyrosine hydroxylase (TH), which acts as a necessary element for transcription. Tyrosine hydroxylase catalyzes the rate-limiting step in the catecholamine biosynthesis and is linked to several common neurological disorders such as Parkinson's and schizophrenia. A 45 nucleotide (nt) sequence (wtTH49) within the human TH promoter contains multiple G-stretches that are extremely well conserved among the primates but deviate in rodents, which raises the possibility of variation in the GQ structures formed in the two orders with the potential for a distinctive functional outcome. Biochemical and biophysical studies, including single-molecule Förster resonance energy transfer, indicate that the wtTH49 sequence can adopt multiple GQ structures by using different combinations of G-stretches. A functional assay performed with 2.8 kb of the 3'-proximal end of the TH promoter and a mutated version (TH49fm; mutated wtTH49) that is unable to form any GQ structure indicates that overall the GQ-enabling wtTH49 sequence is functionally necessary and enhances human TH promoter activity by 5-fold compared to that of the mutant. Two additional mutants, each of which was designed to form distinct GQs, differentially affected reporter gene transcription. A cationic porphyrin TMPyP4 destabilizes the wtTH49 GQ and lowers the level of reporter gene expression, although its analogue, TMPyP2, fails to elicit any response. The 45 nt G-rich sequence within the human TH promoter can form multiple GQ structures, is a necessary element in transcription, and depending on the utilized combination of G-stretches affects transcription in different ways.

Divalent cation-aided identification of physico-chemical properties of metal ions that stabilize RNA G-quadruplexes.

DNA and RNA sequences rich in guanosines (G) can form a four-stranded secondary structure known as a G-quadruplex (GQ), which plays a role in regulation of gene expression at the transcription and translation level. Both DNA and RNA GQs typically use the monovalent K(+) ion for stabilization of the structures. However, the fundamental reasons for K(+) acting as the most stabilizing metal ion for RNA GQs are not known. To identify the properties of a metal ion that stabilizes an RNA GQ we investigated the effect of alkaline earth metal cations and a set of divalent transition metal ions on two previously identified highly stable RNA GQs. Our results based upon circular dichroism and RNase T1 structure mapping data reveal that the RNA GQs are destabilized in the presence of the tested divalent metal cations. The destabilizing effect of a divalent metal cation is reversible upon increasing K(+) concentration. Results show that ionic radius, hydration energy, and binding strength towards the hard ligand (guanine O(6)) are important factors that determine a metal ion's ability to stabilize an RNA GQ. Additionally, the tested set of divalent metal cations incongruously affects RNA and DNA GQs.

Rationally induced RNA:DNA G-quadruplex structures elicit an anticancer effect by inhibiting endogenous eIF-4E expression.

RNA G-quadruplex (GQ) structures act as regulators of a diverse array of cellular processes including translation, pre-mRNA processing, and mRNA targeting. We report here a strategy of harnessing the natural ability of RNA GQs to inhibit translation by rationally inducing a GQ on a targeted mRNA to knockdown endogenous gene expression. We chose to target eIF-4E because of its key role in translation initiation and overexpression in multiple cancers and with the expectation that downregulation of eIF-4E would result in antiproliferation of cancer cells. Targeted hybrid (RNA:DNA) GQ structures were induced at the 5'-untranslated region (UTR) and the protein coding region of the eIF-4E mRNA by rationally designed and partially modified extraneous DNA sequences and their effect on eIF-4E expression was determined. The formation of a stable induced G-quadruplex was established by biophysical and biochemical methods. Thermodynamic parameters calculated from CD melting indicate formation of a stable induced GQ at a physiologically relevant salt concentration. We established the specificity and efficacy of the induced GQ formation by monitoring the targeted repression of a reporter gene. Most importantly we have demonstrated that inducing GQ in the 5'-UTR and the protein coding region of eIF-4E mRNA in human cancer cells results in 30% and 60% inhibition of the endogenous protein expression, respectively. Treating with the GQ inducing oligonucleotide sequences resulted in a decrease in the viability of human cancer cells in a dose-dependent manner. The above concept opens up a new strategy for targeted modulation of endogenous gene expression.

The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells.

We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5'-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D (1)H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9.

Putative G-quadruplex forming sequences (PQS) have been identified in promoter sequences of prominent genes that are implicated among others in cancer and neurological disorders. We explored mechanistic aspects of CRISPR-dCas9-mediated gene expression regulation, which is transient and sequence specific unlike alternative approaches that lack such specificity or create permanent mutations, using the PQS in tyrosine hydroxylase (TH) and c-Myc promoters as model systems. We performed in vitro ensemble and single molecule investigations to study whether G-quadruplex (GQ) structures or dCas9 impede T7 RNA polymerase (RNAP) elongation process and whether orientation of these factors is significant. Our results demonstrate that dCas9 is more likely to block RNAP progression when the non-template strand is targeted. While the GQ in TH promoter was effectively destabilized when the dCas9 target site partially overlapped with the PQS, the c-Myc GQ remained folded and stalled RNAP elongation. We also determined that a minimum separation between the transcription start site and the dCas9 target site is required for effective stalling of RNAP by dCas9. Our study provides significant insights about the factors that impact dCas9-mediated transcription regulation when dCas9 targets the vicinity of sequences that form secondary structures and provides practical guidelines for designing guide RNA sequences.

NAT8L mRNA oxidation is linked to neurodegeneration in multiple sclerosis.

RNA oxidation has been implicated in neurodegeneration, but the underlying mechanism for such effects is unclear. Extensive RNA oxidation occurs within the neurons in multiple sclerosis (MS) brains. Here, we identified selectively oxidized mRNAs in neuronal cells that pertained to neuropathological pathways. N-acetyl aspartate transferase 8 like (NAT8L) is one such transcript, whose translation product enzymatically synthesizes N-acetyl aspartic acid (NAA), a neuronal metabolite important for myelin synthesis. We reasoned that impediment of translation of an oxidized NAT8L mRNA will result in a reduction in its cognate protein, thus lowering the NAA level. This hypothesis is supported by our studies on cells, an animal model, and postmortem human MS brain. Reduced brain NAA level hampers myelin integrity making neuronal axons more susceptible to damage, which contributes to MS neurodegeneration. Overall, this work provides a framework for a mechanistic understanding of the link between RNA oxidation and neurodegeneration.

Decay of Piwi-Interacting RNAs in Human Cells Is Primarily Mediated by 5' to 3' Exoribonucleases.

Piwi-interacting RNAs (piRNAs) are a group of small noncoding RNA molecules that regulate the activity of transposons and control gene expression. The cellular concentration of RNAs is generally maintained by their rates of biogenesis and degradation. Although the biogenesis pathways of piRNAs have been well defined, their degradation mechanism is still unknown. Here, we show that degradation of human piRNAs is mostly dependent on the 5'-3' exoribonuclease pathway. The presence of 3'-end 2'-O-methylation in piRNAs significantly reduced their degradation through the exosome-mediated decay pathway. The accumulation of piRNAs in XRN1 and XRN2 exoribonuclease-depleted cells further supports the 5'-3' exoribonuclease-mediated decay of piRNAs. Moreover, formation of stable secondary structures in piRNAs slows the rate of XRN1-mediated degradation. Our findings establish a framework for the piRNA degradation mechanism in cells and thus provide crucial information about how the basal level concentration of piRNAs is maintained in cells.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5' UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5' UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5' cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5' UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5' cap. In this review, we will focus only on the role of RNA GQs present in the 5' UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.

Direct experimental evidence for quadruplex-quadruplex interaction within the human ILPR.

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPR(n)(=4)). Mobilities of ILPR(n)(=4) in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPR(n)(=4) most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPR(n)(=2)). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPR(n)(=4) at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex-quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPR(n)(=4) that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

The BHMT-betaine methylation pathway epigenetically modulates oligodendrocyte maturation.

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes.

Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single-molecule level.

Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double-stranded DNA. Whereas single-molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C)-rich region complementary to G quadruplex-forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5'-(TGTCCCCACACCCC)(2), a predominate variant in the insulin-linked polymorphic region (ILPR). We have observed two species with the change in contour length (DeltaL) of 10.4 (+/-0.1) and 5.1 (+/-0.5) nm, respectively. Since DeltaL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br(2) footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23% to 1.3%) with pH 5.5 --> 7.0, which is a well-established behavior for i-motifs. In contrast to that of the i-motif, the formation of the second species with DeltaL of 5.1 nm remains unchanged (6.1 +/- 1.6%) in the same pH range, implying that pH-sensitive C:CH(+) pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the DeltaG(unfold) of an i-motif (16.0 +/- 0.8 kcal/mol), the decreased free energy in the partially folded structure (DeltaG(unfold) 10.4 +/- 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH(+) pairs. Both DeltaL and DeltaG(unfold) argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of an i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.

An RNA G-quadruplex is essential for cap-independent translation initiation in human VEGF IRES.

RNA G-quadruplexes located within the 5'-UTR of mRNA are almost always known to be associated with repression of cap-dependent translation. However, in this report we present functional as well as structural evidence that sequence redundancy in a G-rich segment within the 5'-UTR of human VEGF mRNA supports a 'switchable' RNA G-quadruplex structure that is essential for IRES-mediated translation initiation. Additionally, utilization of a specific combination of G-tracts within this segment allows for the conformational switch that implies a tunable regulatory role of the quadruplex structure in translation initiation. A sequence engineered from a functionally handicapped mutant moderately rescued the activity, further indicating the importance of G-quadruplex structure for VEGF IRES-A function. This to our knowledge is the first report of a conformationally flexible RNA G-quadruplex which is essential for IRES-mediated translation initiation.

The role of RNA G-quadruplexes in human diseases and therapeutic strategies.

G-quadruplexes (GQs) are four-stranded secondary structures formed by G-rich nucleic acid sequence(s). DNA GQs are present abundantly in the genome and affect a wide range of processes associated with DNA. Recent studies show that RNA GQs are present in different transcripts, including coding and noncoding areas of mRNA, telomeric RNA as well as in other premature and mature noncoding RNAs. When present at specific locations within the RNAs, GQs play important roles in key biological functions, including the regulation of gene expression and telomere homeostasis. RNA GQs regulate pre-mRNA processing, such as splicing and polyadenylation. Evidently, among other processes, RNA GQs also control mRNA translation, miRNA and piRNA biogenesis, and RNA localization. The regulatory mechanisms controlled by RNA GQs mainly involve binding to RNA binding protein that modulate GQ conformation or serve as an entity in recruiting additional protein regulators to act as a block element to the processing machinery. Here we provide an overview of the ever-increasing number of discoveries revealing the role of RNA GQs in biology and their relevance in human diseases and therapeutics. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA in Disease and Development > RNA in Disease.

An unusually stable G-quadruplex within the 5'-UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cells.

MT3-MMP is a matrix metalloproteinase involved in the regulation of cancer cell invasion and metastasis. The MT3-MMP mRNA contains a 20-nucleotide G-rich region (M3Q) upstream of the initiation codon. Herein, we report that the M3Q purine-only sequence forms an extremely stable intramolecular G-quadruplex structure and has an inhibitory role on translation of a reporter gene in eukaryotic cells. The formation of the G-quadruplex structure was indicated by circular dichroism (CD) spectroscopy and enzymatic footprinting with RNase T1. The unusual stability of the G-quadruplex was evidenced when addition of only 1 mM KCl resulted in about a 30 degrees C increase in the melting temperature (T(m)), as compared to that obtained in the absence of added salt. The T(m) was independent of the RNA concentration, suggesting an intramolecular G-quadruplex structure. Additionally, in a dual luciferase reporter assay performed in eukaryotic cells, the M3Q motif present in the context of the entire 5'-UTR of MT3-MMP repressed activity of its downstream gene by more than half. To the best of our knowledge, the naturally occurring M3Q sequence forms one of the most stable, intramolecular RNA G-quadruplexes reported. This report is the first to establish a functional role of a G-quadruplex forming sequence within the MT3-MMP 5'-UTR in the regulation of translation in eukaryotic cells.

ILPR G-quadruplexes formed in seconds demonstrate high mechanical stabilities.

The insulin linked polymorphism region (ILPR) is known to regulate transcription of the gene coding for insulin. The ILPR has guanine rich segments, suggesting that G quadruplexes may be responsible for this regulatory role. Using mechanical unfolding in a laser tweezers instrument and circular dichroism (CD) spectroscopy, we provide compelling evidence that highly stable parallel and antiparallel G quadruplex structures coexist in the predominant ILPR sequence of (ACAGGGGTGTGGGG)(2) at a physiologically relevant concentration of 100 mM KCl. Experiments at the single molecular level have shown that unfolding forces for parallel and antiparallel structures (F(unfold): 22.6 vs 36.9 pN, respectively) are higher than the stall forces of enzymes having helicase activities. From a mechanical perspective alone, these data support the hypothesis that G quadruplexes may cause replication slippage by blocking replication process. Using the unique combination of the rupture force and the contour length measured by laser tweezers, the simultaneous determination of probable parallel and antiparallel G quadruplex structures in a solution mixture has been achieved. Jarzynski's equality analysis has revealed that the antiparallel G quadruplex is thermodynamically more stable than the parallel conformer (DeltaG (unfold): 23 vs 14 kcal/mol, respectively). On the other hand, kinetic measurements have indicated that both parallel and antiparallel structures fold rather rapidly (k(fold): 0.4 vs 0.3 s(-1), respectively), suggesting that they may be kinetically accessible for gene control. This work provides an unprecedented mechanical perspective on G quadruplex stability, presenting a unique opportunity to predict the functional consequence when motor enzymes encounter such structures.