Draft Genome Sequences of Bacillus sp. Strains RM1(2019), RM2(2019), RM9(2019), RM11(2019), and RM15(2019), Isolated from Temperate Soils in the Hudson River Valley of New York.

We report the draft genome sequences of five novel Bacillus strains isolated from temperate soils in Annandale-on-Hudson, NY. Strains RM1(2019), RM9(2019), and RM15(2019) were identified as Bacillus toyonensis, while RM11(2019) was identified as Bacillus thuringiensis The draft genome of strain RM2(2019) was unclassified and likely represents a new species.

A ruthenium-platinum metal complex that binds to sarcin ricin loop RNA and lowers mRNA expression.

IT127 is a dinuclear transition metal complex that contains a Pt(ii) and a Ru(iii) metal center. We have shown that IT127 is significantly more effective in binding the 29-base sarcin ricin loop (SRL) RNA in comparison to Cisplatin, a hallmark anticancer agent. Binding site analysis shows that IT127 prefers purine bases and the GAGA tetraloop region of SRL RNA. Our results with a dihydrofolate reductase (DHFR) model system reveal that IT127 binding to mRNA reduces translation of DHFR enzyme and that the Ru(iii) and Pt(ii) centers in IT127 appear to work in a synergistic manner.

Synthesis and characterization of water-soluble, heteronuclear ruthenium(III)/ferrocene complexes and their interactions with biomolecules.

The reaction of Na[RuCl4(SO(CH3)2)2], 1, with one equivalent of FcCONHCH2C6H4N (Fc=FeC10H9), L1, FcCOOCH2CH2C3H3N2, L2, FcCOOC6H4N, L3, afforded the dinuclear species, Na[FcCONHCH2C6H4N[RuCl4(SO(CH3)2)]], RuL1, Na[FcCOOCH2CH2C3H3N2[RuCl4(SO(CH3)2)]], RuL2, Na[FcCOOC6H4N(RuCl4(SO(CH3)2))], RuL3, respectively, yielding, in each case, a ferrocene moiety bridged to a ruthenium center. The complexes were characterized by NMR, IR, and XRD (X-ray diffraction). The sulfoxide ligands are bonded to the metal through the sulfur atom. The complexes were evaluated for their biological activity with pBluescript DNA plasmid, and the protein BSA (bovine serum albumin). These reactions were monitored by XAS (X-ray absorption spectroscopy), EXAFS (extended X-ray Absorption Fine Structure), NMR, UV/visible, emission spectroscopy, and gel electrophoresis. Donor atoms from the biomolecules substitute for the chloride ligands in the parent complexes.

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study.

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.

Chemical probing of RNA with the hydroxyl radical at single-atom resolution.

While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5'-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5'-aldehyde. We conclude that hydroxyl radical abstracts a 5'-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5' deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2'-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.

RNA binding and inhibition of primer extension by a Ru(III)/Pt(II) metal complex.

AH197, a trinuclear Ru(III)/Pt(II) metal complex, is strikingly more effective than the hallmark anticancer drug cisplatin and the Ru(III) clinical candidate NAMI-A in its binding to RNA and inhibition of primer DNA synthesis. Heteromultinuclear complexes could potentially serve as far better chemotherapeutics than mononuclear complexes.

Footprinting protein-DNA complexes using the hydroxyl radical.

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.

Addressing the problems of base pairing and strand cyclization in template-directed synthesis: a case for the utility and necessity of 'molecular midwives' and reversible backbone linkages for the origin of proto-RNA.

Nucleic acid synthesis is precisely controlled in living organisms by highly evolved protein enzymes. The remarkable fidelity of information transfer realized between template and product strands is the result of both the spatial selectivity of the polymerase active site for Watson-Crick base pairs at the point of nucleotide coupling and subsequent proof-reading mechanisms. In the absence of naturally derived polymerases, in vitro template-directed synthesis by means of chemically activated mononucleotides has proven remarkably inefficient and error-prone. Nevertheless, the spontaneous emergence of RNA polymers and their protein-free replication is frequently taken as a prerequisite for the hypothetical 'RNA world'. We present two specific difficulties that face the de novo synthesis of RNA-like polymers in a prebiotic (enzyme-free) environment: nucleoside base selection and intramolecular strand cyclization. These two problems are inherent to the assumption that RNA formed de novo from pre-existing, chemically-activated mononucleotides in solution. As a possible resolution to these problems, we present arguments and experimental support for our hypothesis that small molecules (referred to as 'molecular midwives') and alternative backbone linkages (under equilibrium control) facilitated the emergence of the first RNA-like polymers of life.

Assembly of an antiparallel homo-adenine DNA duplex by small-molecule binding.

Molecules that reversibly bind DNA and trigger the formation of non-Watson-Crick secondary structures would be useful in the design of dynamic DNA nanostructures and as potential leads for new therapeutic agents. We demonstrate that coralyne, a small crescent-shaped molecule, promotes the formation of a duplex secondary structure from homo-adenine oligonucleotides. AFM studies reveal that the staggered alignment of homo-adenine oligonucleotides upon coralyne binding produces polymers of micrometers in length, but only 2 nm in height. A DNA duplex was also studied that contained eight A.A mismatches between two flanking 7-bp Watson-Crick helices. CD spectra confirm that the multiple A.A mismatches of this duplex bind coralyne in manner similar to that of homo-adenine oligonucleotides. Furthermore, the melting temperature of this hybrid duplex increases by 13 degrees C upon coralyne binding. These observations illustrate that the helical structure of the homo-adenine-coralyne duplex is compatible with the B-form DNA helix.

Controlling nucleic acid secondary structure by intercalation: effects of DNA strand length on coralyne-driven duplex disproportionation.

Small molecules that intercalate in DNA and RNA are powerful agents for controlling nucleic acid structural transitions. We recently demonstrated that coralyne, a small crescent-shaped molecule, can cause the complete and irreversible disproportionation of duplex poly(dA)*poly(dT) into triplex poly(dA)*poly(dT)*poly(dT) and a poly(dA) self-structure. Both DNA secondary structures that result from duplex disproportionation are stabilized by coralyne intercalation. In the present study, we show that the kinetics and thermodynamics of coralyne-driven duplex disproportionation strongly depend on oligonucleotide length. For example, disproportionation of duplex (dA)16*(dT)16 by coralyne reverts over the course of hours if the sample is maintained at 4 degrees C. Coralyne-disproportioned (dA)32. (dT)(32), on the other hand, only partially reverts to the duplex state over the course of days at the same temperature. Furthermore, the equilibrium state of a (dA)16*(dT)16 sample in the presence of coralyne at room temperature contains three different secondary structures [i.e. duplex, triplex and the (dA)16 self-structure]. Even the well-studied process of triplex stabilization by coralyne binding is found to be a length-dependent phenomenon and more complicated than previously appreciated. Together these observations indicate that at least one secondary structure in our nucleic acid system [i.e. duplex, triplex or (dA)n self-structure] binds coralyne in a length-dependent manner.