Enhanced Tetrahedral Ordering of Water Molecules in Minor Grooves of DNA: Relative Role of DNA Rigidity, Nanoconfinement, and Surface Specific Interactions

Confinement and Surface specific interactions call induce Structures otherwise unstable at that temperature and pressure. Here we Study the groove specific water dynamics ill the nucleic acid sequences, poly-AT and poly-GC, in long B-DNA duplex chains by large scale atomistic molecular dynamics simulations, accompanied by thermodynamic analysis. While water dynamics in the major groove remains insensitive to the sequence differences, exactly the opposite is true for the minor groove water. Much slower water dynamics observed in the minor grooves (especially in the AT minor) call be attributed to all enhanced tetrahedral ordering (< t(h)>) of water. The largest value of < t(h)> in the AT minor groove is related to the spine of hydration found in X-ray Structure. The calculated configurational entropy (S-C) of the water molecules is found to be correlated with the self-diffusion coefficient of water in different region via Adam-Gibbs relation D = A exp(-B/TSC), and also with < t(h)>.

Molecular markers for discriminating Streptococcus pyogenes and S.dysgalactiae subspecies equisimilis

Given the increasing aetiological importance of Streptococcus dysgalactiae subspecies equisimilis in diseases which are primarily attributed to S. pyogenes, molecular markers are essential to distinguish these species and delineate their epidemiology more precisely. Many clinical microbiology laboratories rely on agglutination reactivity and biochemical tests to distinguish them. These methods have limitations which are particularly exacerbated when isolates with mixed properties are encountered. In order to provide additional distinguishing parameters that could be used to unequivocally discriminate these two common pathogens, we assess here three molecular targets: the speB gene, intergenic region upstream of the scpG gene (IRSG) and virPCR. Of these, the former two respectively gave positive and negative results for S. pyogenes, and negative and positive results for S. dysgalactiae subsp. equisimilis. Thus,a concerted use of these nucleic acid-based methods is particularly helpful in epidemiological surveillance to accurately assess the relative contribution of these species to streptococcal infections and diseases.

Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Lyophilized disaccharides – the effect of water during storage

Nearly one fourth of new medicinal molecules are biopharmaceutical (protein, antibody or nucleic acid derivative) based. However, the administration of these compounds is not always that straightforward due to the fragile nature of aforementioned domains in GI-tract. In addition, these molecules often exhibit poor bioavailability when administered orally. As a result, parenteral administration is commonly preferred. In addition, shelf-life of these molecules in aqueous environments is poor, unless stored in low temperatures. Another approach is to bring these molecules to anhydrous form via lyophilization resulting in enhanced stability during storage. Proteins cannot most commonly be freeze dried by themselves so some kind of excipients are nearly always necessary. Disaccharides are commonly utilized excipients in freeze-dried formulations since they provide a rigid glassy matrix to maintain the native conformation of the protein domain. They also act as "sink"-agents, which basically mean that they can absorb some moisture from the environment and still help to protect the API itself to retain its activity and therefore offer a way to robust formulation. The aim of the present study was to investigate how four amorphous disaccharides (cellobiose, melibiose, sucrose and trehalose) behave when they are brought to different relative humidity levels. At first, solutions of each disaccharide were prepared, filled into scintillation vials and freeze dried. Initial information on how the moisture induced transformations take place, the lyophilized amorphous disaccharide cakes were placed in vacuum desiccators containing different relative humidity levels for defined period, after which selected analyzing methods were utilized to further examine the occurred transformations. Affinity to crystallization, water sorption of the disaccharides, the effect of moisture on glass transition and crystallization temperature were studied. In addition FT-IR microscopy was utilized to map the moisture distribution on a piece of lyophilized cake. Observations made during the experiments backed up the data mentioned in a previous study: melibiose and trehalose were shown to be superior over sucrose and cellobiose what comes to the ability to withstand elevated humidity and temperature, and to avoid crystallization with pharmaceutically relevant moisture contents. The difference was made evident with every utilized analyzing method. In addition, melibiose showed interesting anomalies during DVS runs, which were absent with other amorphous disaccharides. Particularly fascinating was the observation made with polarized light microscope, which revealed a possible small-scale crystallization that cannot be observed with XRPD. As a result, a suggestion can safely be made that a robust formulation is most likely obtained by utilizing either melibiose or trehalose as a stabilizing agent for biopharmaceutical freeze-dried formulations. On the other hand, more experiments should be conducted to obtain more accurate information on why these disaccharides have better tolerance for elevating humidities than others.

Kohdennetut nanopartikkelit ja isotooppikuvantaminen syövän hoidossa

Syövän diagnostiikassa ja hoidossa nanopartikkelit voivat toimia kuljetinaineina lääke- ja diagnostisille aineille tai nukleiinihappojaksoille. Kantaja-aineeseen voidaan liittää kohdennusmolekyylejä partikkelien passiivista tai aktiivista kohdennusta varten tai radioleima kuvantamista tai radioterapiaa varten. Kantaja-aineiden avulla voidaan parantaa lääkeaineen fysikaalis-kemiallisia ominaisuuksia ja biologista hyötyosuutta, vähentää systeemisiä sivuvaikutuksia, pidentää lääkeaineen puoliintumisaikaa ja siten harventaa annosteluväliä, sekä parantaa lääkeaineen pääsyä kohdekudokseen. Näin voidaan parantaa kemo- ja radioterapian tehoa ja hoidon onnistumisen todennäköisyyttä. Kirjallisuuskatsauksessa perehdytään nanokantajien rooliin syövän hoidossa. Vuosikymmeniä jatkuneesta tutkimuksesta huolimatta vain kaksi (Eurooppa) tai kolme (Yhdysvallat) nanopartikkeliformulaatiota on hyväksytty markkinoille syövän hoidossa. Ongelmina ovat riittämätön hakeutuminen kohdekudokseen, immunogeenisyys ja nanopartikkelien labiilius. Kokeellisessa osassa tutkitaan in vitro ja hiirillä in vivo 99mTc-leimattujen, PEG-verhoiltujen biotiiniliposomien kaksivaiheista kohdennusta ihmisen munasarjan adenokarsinoomasoluihin. Kohdentamiseen käytetään biotinyloitua setuksimabi-(Erbitux®) vasta-ainetta, joka sitoutuu solujen yli-ilmentämiin EGF-reseptoreihin. Kaksivaiheista kohdennusta verrataan suoraan ja/tai passiiviseen kohdennukseen. Tehokkaampien kuvantamismenetelmien kehitys on vauhdittanut kohdennettujen nanopartikkelien tutkimusta. Isotooppikuvantamista käyttäen pystytään seuraamaan radioleiman jakautumista elimistössä ja kuvantamaan solutasolla tapahtuvia ilmiöitä. Kirjallisuuskatsauksessa perehdytään SPECT- ja PET-kuvantamiseen syövän hoidossa, sekä niiden hyödyntämiseen lääkekehityksessä nanopartikkelien kuvantamisessa. Kyseiset kuvantamismenetelmät erottuvat muista menetelmistä korkean erotuskyvyn, herkkyyden ja helppokäyttöisyyden suhteen. Kokeellisessa osassa 99mTc-leimattujen liposomien distribuutiota hiirissä tutkittiin SPECT-CT-laitteen avulla. Aktiivisuus kasvaimessa, pernassa ja maksassa kvantifioitiin InVivoScope-ohjelman ja gammalaskijan avulla. Tuloksia verrattiin keskenään. In vitro-kokeessa saavutettiin kaksivaiheisella kohdennuksella 2,7- 3,5-kertainen (solulinjasta riippuen) hakeutuminen soluihin kontrolliliposomeihin verrattuna. Kuitenkin suora kohdennus toimi kaksivaiheista kohdennusta paremmin in vitro. In vivo –kokeissa liposomit jakautuivat kasvaimeen tehokkaammin i.p.-annosteltuna kuin i.v.-annosteltuna. Kaksivaiheisella kohdennuksella saavutettiin 1,24-kertainen jakautuminen kasvaimeen (% ID/g kudosta) passiivisesti kohdennettuihin liposomeihin verrattuna. %ID/elin oli kohdennetuilla liposomeilla 5,9 % ja passiivisesti kohdennetuilla 5,4%. Todellinen ero oli siis pieni. InVivoScope:n ja gammalaskijan tulokset eivät korreloineet keskenään. Lisätutkimuksia ja menetelmän optimointia vaaditaan liposomien kohdennuksessa kasvaimeen.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Primary structure of sesbania mosaic virus coat protein: its implications to the assembly and architecture of the virus

Sesbania mosaic virus (SMV) is a plant virus that infects Sesbania grandiflora plants in Andhra Pradesh, India. The amino acid sequence of the coat protein of SMV was determined using purified peptides generated by cleavage with trypsin, chymotrypsin, V8 protease and clostripain. The 230 residues so far determined were compared to the corresponding residues of southern bean mosaic virus (SBMV), the type member of sobemoviruses. The overall identity between the sequences is 61.7%. The amino terminal 64 residues, which constitute an independent domain (R-domain) known to interact with RNA, are conserved to a lower extent (52.5%). Comparison of the positively charged residues in this domain suggests that the RNA-protein interactions are considerably weaker in SMV. The residues that constitute the major domain of the coat protein, the surface domain (S-domain, residues 65-260), are better conserved (66.5%). The positively charged residues of this domain that face the nucleic acid are well conserved. The longest conserved stretch of residues (131-142) corresponds to the loop involved in intersubunit interactions between subunits related by the quasi 3-fold symmetry. A unique cation binding site located on the quasi 3-fold axis contributes to the stability of SMV. These differences are reflected in the increased stability of the SMV coat protein and its ability to be reconstituted with RNA at pH 7.5. A major epitope was identified using monoclonal antibodies to SMV in the segment 201-223 which contains an exposed helix in the capsid structure. This region is highly conserved between SMV and SBMV (70%) suggesting that it could represent the site of an important function such as vector recognition.

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

BSSB: BLAST Server for Structural Biologists

The Basic Local Alignment Search Tool (BLAST) is one of the most widely used sequence alignment programs with which similarity searches, for both protein and nucleic acid sequences, can be performed against large databases at high speed. A large number of tools exist for processing BLAST output, but none of them provide three-dimensional structure visualization. This shortcoming has been addressed in the proposed tool BLAST Server for Structural Biologists (BSSB), which maps a BLAST output onto the three-dimensional structure of the subject protein. The three-dimensional structure of the subject protein is represented using a three-color coding scheme (identical: red; similar: yellow; and mismatch: white) based on the pairwise alignment obtained. Thus, the user will be able to visualize a possible three-dimensional structure for the query protein sequence. This information can be used to gain a deeper insight into the sequence-structure correlation. Furthermore, the additional structure-level information enables the user to make coherent and logical decisions regarding the type of input model structure or fragment that can be used for molecular replacement calculations. This tool is freely available to all users at http://bioserver1.physics.iisc.ernet.in/bssb/.

Mutagenic significance of proton acidities in methylated guanine and thymine bases and deoxynucleosides: A theoretical study

Proton changes have been advanced as being the key molecular basis for the mutagenecity of alkylated DNA bases and nucleosides, leading to questions as to which protons are involved and whether the protic changes are tautomeric shifts or abstractions. This semiempirical molecular orbital study seeks to clarify the issue by examining the various possibilities open for these protic changes in a number of methylated guanines and thymines and their deoxynucleosides. Proton shifts leading to tautomer formation are not predicted as being thermodynamically favourable in most cases. The most feasible proton abstractions are predicted to involve the Watson-Crick protons in all cases, which corroborates Watson-Crick proton loss as providing the key molecular basis for the induction of point mutations. The calculated proton acidities correlate well with experimental data. The gas-phase deprotonation enthalpies for a number of alkylated nucleosides are found to correlate linearly with the solvent-phase pK(a) values. The theoretically calculated enthalpies in a simulated aqueous solvent phase of the deprotonation reactions of various nucleic acid bases are also found to have good linear correlations with experimental pK(a) values. The consensus of these calculations is that O-6-alkyldeoxyguanosines, and O-2- and O-4-alkyldeoxythymidines would be mutagenic while N-7-alkyldeoxyguanosines would not be mutagenic (as experiment indicates). The untested N-3-methyldeoxyguanosine is predicted to be mutagenic. (C) 1997 Elsevier Science B.V.

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved.

Translocation and encapsulation of siRNA inside carbon nanotubes

We report spontaneous translocation of small interfering RNA (siRNA) inside carbon nanotubes (CNTs) of various diameters and chirality using all atom molecular dynamics simulations with explicit solvent. We use umbrella sampling method to calculate the free energy landscape of the siRNA entry and translocation event. Free energy profiles show that siRNA gains free energy while translocating inside CNT, and barrier for siRNA exit from CNT ranges from 40 to 110 kcal/mol depending on CNT chirality and salt concentration. The translocation time tau decreases with the increase of CNT diameter with a critical diameter of 24 angstrom for the translocation. In contrast, double strand DNA of the same sequence does not translocate inside CNT due to large free energy barrier for the translocation. This study helps in understanding the nucleic acid transport through nanopores at microscopic level and may help designing carbon nanotube based sensor for siRNA. (C) 2013 American Institute of Physics. http://dx.doi.org/10.1063/1.4773302]

Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

The Non-catalytic ``Cap Domain'' of a Mycobacterial Metallophosphoesterase Regulates Its Expression and Localization in the Cell

Despite highly conserved core catalytic domains, members of the metallophosphoesterase (MPE) superfamily perform diverse and crucial functions ranging from nucleotide and nucleic acid metabolism to phospholipid hydrolysis. Unique structural elements outside of the catalytic core called ``cap domains'' are thought to provide specialization to these enzymes; however, no directed study has been performed to substantiate this. The cap domain of Rv0805, an MPE from Mycobacterium tuberculosis, is located C-terminal to its catalytic domain and is dispensable for the catalytic activity of this enzyme in vitro. We show here that this C-terminal extension (CTE) mediates in vivo localization of the protein to the cell membrane and cell wall as well as modulates expression levels of Rv0805 in mycobacteria. We also demonstrate that Rv0805 interacts with the cell wall of mycobacteria, possibly with the mycolyl-arabinogalactan-peptidoglycan complex, by virtue of its C terminus, a hitherto unknown property of this MPE. Using a panel of mutant proteins, we identify interactions between active site residues of Rv0805 and the CTE that determine its association with the cell wall. Finally, we show that Rv0805 and a truncated mutant devoid of the CTE produce different phenotypic effects when expressed in mycobacteria. Our study thus provides a detailed dissection of the functions of the cap domain of an MPE and suggests that the repertoire of cellular functions of MPEs cannot be understood without exploring the modulatory effects of these subdomains.

Raman Spectroscopic Studies on Screening of Myopathies

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld1 (up1), heldup(2) (hdp(2)), myosin heavy chain7 (Mhc7), actin88F(KM88) (Act88F(KM88)), upheld101 (up101), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and beta-sheet and/or random coil protein content along with a decrease in a-helix protein. Interestingly, the 12th day samples of up1 and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up1 and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up101 and hdp2 (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up1). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.