For many applications it would be desirable to able control the activity of proteins by using an external signal. In present study, we have explored possibility modulating a restriction enzyme with light. By cross-linking two suitably located cysteine residues bifunctional azobenzene derivative, which can adopt cis - or trans -configuration when illuminated UV blue light, respectively, enzymatic controlled in reversible manner. To determine cross-linked show largest “photoswitch effect,”...

2'-Deoxyoligonucleotides and 2'-O-methyloligoribonucleotides carrying one or more 2'-aldehyde groups were synthesized coupled to peptides containing an N-terminal cysteine, aminooxy, hydrazide group give peptide-oligonucleotide conjugates incorporating single multiple in good yield. The facile conjugation method allows specific coupling aqueous solution of unprotected oligonucleotides aldehyde N-terminally modified other small molecules. A 12-mer 2'-O-methyloligoribonucleotide complementary...

In molecular biology, the production of proteins can be effectively inhibited by introducing specific oligonucleotides into a living cell (gene silencing or antisense strategy; important for gene therapy). Calcium phosphate nanoparticles serve as carriers biomolecules in such therapeutic applications due to their high biocompatibility and biodegradability. Stable colloids were prepared coating inorganic with single- double-stranded oligonucleotides. The dispersions analysed dynamic light...

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by action BrCN was developed. A rational design linear precursor studied, and effect factors such as oligonucleotide concentration oligomer-template length ratio evaluated. The efficiency circularization shown to correlate well with secondary structure oligomer (as predicted a simple computer analysis), hairpinlike structures bearing free termini clearly favouring reaction. novel idea,...

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine.It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating normally inert C5 position.We show here substitutions in E.coll methylase M.EcoRIl with either serine or tryptophan results complete loss ability to transfer methyl groups DNA.Interestingly, mutants glycine substitution bind tightly substrate DNA.These resemble wild-type enzyme their binding is not...

Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ(A)-RNAP in Bacillus subtilis) regulate transcription a growth phase-dependent manner. B. subtilis expresses two RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show vitro that shares hallmark features RNA: Both (1) are able serve as templates for pRNA transcription; (2) comparable affinity σ(A)-RNAP; (3) specifically inhibit from DNA promoters, (4) can form stable RNA:pRNA hybrid structures (5) abolish binding...

Here we investigated the ability of human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped substrates with damage located a defined position template strand. Maximum velocities Michaelis constant values were determined study synthesis presence either Mg(2+) or Mn(2+). Additionally, influence hRPA (human replication protein A) hPCNA proliferating cell nuclear antigen) on TLS (translesion synthesis) activity was examined. The results show that (i) polymerase is...

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage part of multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored repair opportunity apurinic/apyrimidinic (AP site) composed clustered lesion with 5-formyluracil (5-foU) by base excision (BER) proteins. We found, that if AP is shifted relative to 5-foU opposite strand, it could be repaired primarily via short-patch BER pathway. In this case, cleavage...

6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to polymerase holoenzymes. The mechanism of action was investigated large extent E. coli, however, lack (ΔssrS) demonstrated be unfavorable but not essential for cell survival under various growth conditions. In the study, we revealed, first time, lethal phenotype ΔssrS strain presence high concentrations H2O2. This rescued by complementation ssrS gene on plasmid. We performed comparative...

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize G-quadruplex (G4) structures, which, along with regulatory functions, have negative impact on genome integrity. Here, we studied effect G4 DNA-binding activity MutS from Rhodobacter sphaeroides (methyl-independent MMR) comparison Escherichia coli (methyl-directed and evaluated influence functioning other proteins involved initial steps MMR. For...

An efficient method for synthesis of oligonucleotide 5′-conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5′-carboxylic acid function were obtained by use novel non-nucleosidic phosphoramidite building block, where the carboxylic moiety was protected 2-chlorotrityl group. The protecting group stable to coupling conditions used in solid-phase assembly, but easily deprotected mild acidic treatment. may be removed also ammonolysis....

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular approach is based on modification R.SsoII with azobenzene derivatives, which allows DNA binding cleavage via illumination light. variants single cysteine residues introduced at selected positions were obtained modified maleimidoazobenzene derivatives. twofold change in enzymatic...

An efficient method for synthesis of oligonucleotide 2′-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2′-O-carboxymethyl group were obtained by use novel uridine 3′-phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting stable to conditions used in solid-phase assembly, but easily removed Pd(0) and morpholine treatment. 2′-O-Carboxymethylated then efficiently conjugated support under...

The double‐stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation DNA methyltransferase Sso II to sequence‐specific decoys was demonstrated. obtained results assume that synthetic bearing a new site can be as tools study and manipulate biological systems.

Interaction of the cationic surfactants benzalkonium chloride and 1-hexadecylpyridinium chloride, in concentration range 0.1 microM to 1 mM with calf thymus DNA short 19-mer double-stranded has been examined solution using UV absorption fluorescent spectroscopies at liquid-solution interface by thickness-shear mode acoustic wave sensor. Higher concentrations surfactant resulted an increase absorption, decrease melting temperature van't Hoff enthalpy DNA. Both induce fluorescence quenching...

Oligonucleotide‐peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling pre‐synthesized peptides 2′‐position selected nucleotide an otherwise protected oligonucleotide chain attached solid support.

Abstract Chemical syntheses of 2′-O-(allyloxycarbonyl)methyladenosine, 2′-O-(methoxycarbonyl)methyladenosine and 2′-O-(2,3-dibenzoyloxy)propyluridine 3′-2-cyanoethyl-N,N-diisopropyl phosphoramidite building blocks are described. These monomers were used successfully to incorporate carboxylic acid, 1,2-diol aldehyde functionalities into synthetic oligonucleotides.