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Biomedical subjects

Shankar Balasubramanian

Publications and source records attributed to Shankar Balasubramanian.

At least 19 recordsLinked to original sources
PubMed2004

Loop-length-dependent folding of G-quadruplexes.

Guanine-rich DNA sequences can form a large number of structurally diverse quadruplexes. These vary in terms of strand polarity, loop composition, and conformation. We have derived guidelines for understanding the influence of loop length on the structure adopted by intramolecular quadruplex-forming sequences, using a combination of experimental (using CD and UV melting data) and molecular modeling and simulation techniques. We find that a parallel-stranded intramolecular quadruplex structure is the only possible fold when three single residue loops are present. When single thymine loops are present in combination with longer length loops, or when all loops are longer than two residues, both parallel- and antiparallel-folded structures are able to form. Multiple conformations of each structure are likely to coexist in solution, as they were calculated to have very similar free energies.

Base Sequence↗
PubMed2004

Inhibition of human telomerase activity by an engineered zinc finger protein that binds G-quadruplexes.

The G-quadruplex nucleic acid structural motif is a target for designing molecules that could potentially modulate telomere length or have anticancer properties. We have recently described an engineered zinc finger protein (Gq1) that binds with specificity to the intramolecular G-quadruplex formed by the human telomeric sequence 5'-(GGTTAG)(5)-3' (Isalan et al. (2001) Biochemistry 40, 830-836). Here, we report that Gq1 is able to arrest the action of a DNA polymerase on a template-containing telomeric sequence. Inhibition occurs in a concentration-dependent manner, probably by forming a stabilized G-quadruplex.protein complex. Furthermore, Gq1 inhibits the apparent activity of the enzyme telomerase in vitro, with an IC(50) value of 74.3 +/- 11.1 nM. Possible molecular mechanisms of inhibition are discussed, together with the potential for using engineered zinc fingers to interfere with the cellular processes associated with telomere function.

DNA↗
PubMed2004

Measuring single-molecule nucleic acid dynamics in solution by two-color filtered ratiometric fluorescence correlation spectroscopy.

This work presents a general method for determining single-molecule intramolecular dynamics in biomolecules by using a reporter fluorophore, whose fluorescence is quenched or partially quenched as a result of intramolecular motion, and a remote observer fluorophore. These fluorophores were excited independently with two different lasers, and the ratio of the two fluorophores' fluorescence was calculated. The time-varying ratio was then filtered to reduce contributions from molecules outside the overlapped laser volume and then correlated. The rates of opening and closing of a DNA hairpin were measured by using both fluorescence correlation spectroscopy and this method for comparison. We found at 50 pM, where molecules were studied one by one as they diffused through the probe volume, we obtained accurate opening and closing rates and could also measure dynamic heterogeneity. To demonstrate applicability to a more complex biological molecule we then probed intramolecular motions in the dimer of a human telomerase RNA fragment (hTR(380-444)), in the presence of an excess of monomer. The motion was found to occur on the time scale of 180-750 micros and slowed with increasing magnesium ion concentration. Blocking experiments using complementary oligonucleotides suggested that the motion involves substantial changes in dimer tertiary structure. This method appears to be a general method for selectively studying intramolecular motion in large biomolecules or complexes.

Base Sequence↗
PubMed2004

Tetrapeptides induce selective recognition for G-quadruplexes when conjugated to a DNA-binding platform.

3,6-Bis-peptide acridine and acridone conjugates have been designed and synthesised to selectively interact with G-quadruplex DNA. The ligand properties are peptide sequence dependent, the highest discrimination being obtained with the FRHR tetrapeptide (up to >50-fold specificity). Molecular modeling studies have helped us rationalise the data and suggest that human telomeric quadruplex DNA can readily accommodate tetrapeptides, and furthermore that FRHR contributes to stabilization of the complex by non-bonded interactions within the TTA loop pockets of the quadruplex. These studies indicate that targeting distinct features of a G-quadruplex with hybrid molecules is a promising strategy for discriminating between quadruplex and duplex DNA.

Acridines↗
PubMed2004

Formation of an interlocked quadruplex dimer by d(GGGT).

A tetranucleotide sequence d(GGGT) has been shown to self-assemble into an interlocking quadruplex dimer. UV-melting studies indicated the existence of two species that each showed distinct quadruplex melting transitions, a low-T(m) species, Q(l), and a high-T(m) species, Q(h). Conditions were controlled to favor the formation of either Q(l) or Q(h). Q(l) and Q(h) each showed circular dichroism spectra characteristic of parallel quadruplexes. Negative ion nano-electrospray ionization mass spectrometry confirmed that Q(l) was a tetrameric complex, d(GGGT)(4), and Q(h) was an octameric complex, d(GGGT)(8). High-resolution (1)H NMR spectroscopy evidenced that d(GGGT)(4) was a C(4)-symmetric parallel tetramolecular quadruplex. The (1)H NMR spectrum of d(GGGT)(8) was consistent with a structure formed by the dimerization of a parallel, "slipped" tetramolecular quadruplex that has its diagonal strands staggered by one base. This "slippage" results in two guanine bases at the 5' end of the quadruplex being presented diagonally that are not involved in tetrads. Two such "slipped" quadruplexes dimerize via these free G-bases at the 5' ends by forming an extra G-tetrad. Each "slipped" quadruplex contributes two guanine bases to this extra G-tetrad. The formation of a novel GTGT tetrad is also observed at both the 3' ends of the interlocked quadruplex dimer.

Circular Dichroism↗
PubMed2004

Studies on the chemical stability and synthetic utility of an oxazolidine linker for solid-phase chemistry.

A chemical stability study on the oxazolidine linker system has been carried out using a dual-linker analytical construct within a parallel reaction scan. The study established the compatibility of the oxazolidine platform with a wide range of commonly employed synthetic reaction conditions including nucleophilic, oxidizing, and reducing conditions. The scan was further used to probe and optimize acidic conditions under which the oxazolidine could release the substrate from the solid-support and to identify reagents that could cleave while retaining other acid-labile groups. The solid-phase synthesis of a small molecular array established the utility of oxazolidine aldehyde 1 as a building block for asymmetric chemistry while exploiting the data generated by the reaction scan.

Journal Article↗
PubMed2004

Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution.

We have used two-color fluorescence coincidence detection to directly count individual protein-antibody complexes of protein G or herpes simplex virus labeled with one or more red- and blue-excited antibodies. This allowed quantitative measurement of the concentration of the protein-antibody complexes over 3 orders of magnitude down to the femtomolar level. Single molecule measurements in diluted serum are also possible. The sample preparation is simple, takes place in solution, and requires no separation. Both the antibody affinity and complex dissociation rate are important in determining the sensitivity of the method. At present, the sensitivity limit of 50 fM is determined by the encounter rate of the labeled analyte with the probe volume. This method can be used to detect and quantitate proteins and to measure the stoichiometry, equilibrium constant, and dissociation rate of protein-protein complexes at low concentrations.

Animals↗
PubMed2004

A PNA4 quadruplex.

A tetrameric PNA, TGGG, has been shown to form an intermolecular G-quadruplex. Nanoelectrospray mass spectrometry, combined with solution-phase H/D exchange, established formation of a specific tetramolecular complex. UV melting studies show that this complex undergoes a quadruplex melting transition. This is a novel four-stranded structure that offers the gross structural features of a DNA quadruplex, but without the negatively charged backbone.

Chemical Phenomena↗
PubMed2004

Targeting the single-strand G-rich overhang of telomeres with PNA inhibits cell growth and induces apoptosis of human immortal cells.

Telomeres are believed to stabilize chromosomes through several mechanisms that are dependent upon specific DNA-DNA and protein-DNA interactions. Telomeres are maintained by the enzyme telomerase. Telomerase activity, which is below detectable level in almost all types of diploid cells, is re-activated in most immortal and cancer cells. For this study, we designed peptide nucleic acid (PNA) oligonucleotides targeted to the telomeric G-rich strand, and tested their efficacy to reverse the immortality of transformed human fibroblasts. Anti-telomere PNAs, transfected into human fibroblasts along with a selectable marker, resulted in a significant reduction in colony size and elicited cell death by apoptosis. This PNA inhibitor does not inhibit telomerase activity in vitro, suggesting a distinct cellular mechanism from known PNA inhibitors. A combination of this class of PNA inhibitor with a PNA that does block telomerase activity resulted in nearly complete inhibition of colony growth, induction of apoptosis, and an apparent reduction in telomere length. Each effect was greater than that evoked by either agent alone, indicating enhanced efficacy for therapeutic approaches that target multiple, distinct mechanism of telomere maintenance.

Adenocarcinoma↗
PubMed2003

Studies on the structure and dynamics of the human telomeric G quadruplex by single-molecule fluorescence resonance energy transfer.

We have investigated the structure and unfolding kinetics of the human telomeric intramolecular G quadruplex by using single-molecule fluorescence resonance energy transfer. An exploration of conformational heterogeneity revealed two stable folded conformations, in both sodium- and potassium-containing buffers, with small differences between their enthalpies and entropies. Both folded conformations can be opened by the addition of a 21-base complementary DNA oligonucleotide. The unfolding of both substates occurs at the same rate, which showed dependence on the monovalent metal cation present. Temperature-dependence studies in 100 mM KCl gave an apparent activation enthalpy and entropy of 6.4 +/- 0.4 kcal.mol-1 and -52.3 +/- 1.4 cal.mol-1.K-1, respectively, indicating that the unfolding is entropically driven and can occur easily. In contrast, in 100 mM NaCl the respective values are 14.9 +/- 0.2 kcal.mol-1 and -23.0 +/- 0.8 cal.mol-1.K-1, suggesting a more significant enthalpic barrier. Molecular modeling suggests that the two species are likely to be the parallel and antiparallel quadruplex structures. The unfolding free energy barrier is estimated to be between 3 and 15 kBT based on Kramers' theory. We conclude that under near-physiological conditions these structures coexist and can interconvert on a minute time scale.

Base Composition↗
PubMed2003

Identification of a new RNA.RNA interaction site for human telomerase RNA (hTR): structural implications for hTR accumulation and a dyskeratosis congenita point mutation.

The enzyme telomerase is a ribonucleoprotein that has a critical role in the maintenance of stable telomeres in organisms that possess linear chromosomes. Using a recently developed single molecule fluorescence coincidence method, we have studied the RNA component of telomerase (hTR) and directly observed multimerisation of hTR in solution. RNA mutagenesis and blocking oligonucleotides were employed to identify the single-stranded internal loop J7b/8a as an important and specific hTR.hTR interaction site. This observation was confirmed by studies on a model RNA fragment (hTR(380-444)), comprising part of the H/ACA domain, the internal loop J7b/8a and the CR7 domain, that was found to dimerise. Substitution mutagenesis within the proposed RNA.RNA interaction site of hTR(380-444) resulted in a loss of dimerisation potential and insertion of the dyskeratosis congenita mutation C408G led to a significant reduction in dimer formation. Together, these results suggest that this RNA.RNA interaction site may be functionally relevant.

Binding Sites↗
PubMed2003

G-quadruplex-specific peptide-hemicyanine ligands by partial combinatorial selection.

G-quadruplex DNA-specific ligands were generated using a combinatorial selection of tetrapeptides which were then conjugated to a hemicyanine scaffold. The heterocycle enhanced peptide binding affinity by approximately 1000-fold to give ligands with near micromolar affinity and >40-fold discrimination for quadruplex DNA over duplex.

Carbocyanines↗
PubMed2003

Kinetics of unfolding the human telomeric DNA quadruplex using a PNA trap.

The kinetics of opening of the DNA quadruplex formed by the human telomeric repeat have been investigated using real-time fluorescence resonance energy transfer (FRET) measurements with a peptide nucleic acid (PNA) trap. It has been found that this opening is zero-order with respect to PNA, indicating that the initial step is a rate-limiting internal rearrangement of the quadruplex. A study of the temperature dependence of the rate of quadruplex opening was performed and the activation energy of the process estimated to be 98 +/- 8 kJ mol(-1).

Circular Dichroism↗
PubMed2003

Ultrasensitive coincidence fluorescence detection of single DNA molecules.

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.

DNA↗