Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics.

BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

The structural diversity of C-rich DNA aggregates: unusual self-assembly of beetle-like nanostructures.

We studied the ability of oligonucleotides CnT25 (n = 2, 5, 7, 9, 12, 25) to form an intermolecular i-motif using circular dichroism, ultra-violet spectroscopy, nuclear magnetic resonance, high-resolution atomic force microscopy, high-performance liquid chromatography, and molecular dynamics simulations. The arrangement of single-stranded oligonucleotides in multimer i-motifs was very unusual: C-tracts of different oligonucleotides followed each other consecutively in order to fold into a closed intermolecular i-motif core with minimal loops (one cytidine in a loop spanning over a minor groove, three cytidines in a loop over a major groove); intact T-tracts protruded from predefined loci allowing visualization of beetle-like nanostructures by atomic force microscopy. The same structures were formed from analogous biotinylated oligonucleotides demonstrating one of the potential applications of such structures as carriers of multiple functional groups. Our findings open up possibilities for the rational design of pH-sensitive DNA aggregates and evaluation of the efficiency of their assembly.

[Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. II. potentsial'naia rol' G-kvadrupleksnykh struktur v genomnykh perestroikakh.

Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

[Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. I. Nekanonicheskie struktury.

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

[G-quadruplex ligands: mechanisms of anticancer action and target binding].

Since the discovery of potential therapeutic value of quadruplex secondary nucleic acids structures, many compounds that stabilize these targets were found. Such progress became possible due to understanding of the structural features of G-quadruplexes. Quadruplex ligands selectively suppress the growth of tumor cells by indirect inhibition of the telomerase activity and/or attenuation of oncogenes' overexpression. Therapeutic effect demonstrated in vivo supports the prospect of such compounds for the development of the targeted anticancer drugs. This review reveals the significance of G-quadruplexes as therapeutic targets and focuses on biochemical properties of the low molecular weight quadruplex ligands.

[Oligonucleotides with peptide internucleotide linkage. A novel class of modified oligonucleotides].

Oligonucleotide analogues with replacement of one or more internucleotide phosphodiester linkages with glycine, L- and D-alanine residues have been synthesized (C3'-NH-C(O)-CH(X)-NH-C(O)-C4', X = H, (S)-CH3 and (R)-CH3). The stability of the duplexes formed by the modified oligonucleotides and their wild-type complements have been studied. Incorporation of glycine and L-alanine residues have been shown to substantially decrease the stability the modified duplexes in comparison with that of the wild-type ones (DeltaT(m) approximately -2 degrees C per modification), while the analogs with D-alanine-containing linkages appeared to form duplexes with increased stability (DeltaT(m), approximately +0.2 degrees C per modification).

[Oligonucleotide analogues containing a C3'-NH-C(O)-CH(2)-C5' amide internucleotide bond].

A dinucleoside containing a C3'-NH-C(O)-CH(2)-C5' amide internucleoside bond was synthesized by the interaction of 3'-deoxy-3'-amino-5'-O-(tert-butyldimethylsilyl)thymidine with 3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-5'-deoxy-5'-carboxymethylribosylthymine, which was obtained from 2'-O-acetyl-3'-O-benzyl-5'-deoxy-5'-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation and ester saponification. After standard manipulation with protecting groups, the dinucleoside was converted into 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones.

[Synthesis of 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides, the precursors of oligonucleotides with the amide internucleoside bond C3'-NH-C(O)-CH2-C5'].

A method for the synthesis of 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides has been developed. 3-O-Benzyloxymethyl-1,2-O-isopropylidene-alpha-D-allofuranose was oxidized by sodium periodate to form a 5-aldo derivative, which was converted by the reaction with triethylphosphonoacetate in the presence of sodium hydride into a 5-deoxy-5-ethoxycarbonylmethylene derivative. The hydration of the unsaturated compound gave 5-deoxy-5-ethoxycarbonylmethyl-l,2-O-isopropylidene-alpha-D-ribofuranose. After the benzylation of 3-hydroxyl, the removal of the isopropylidene group by heating with acetic acid, and the subsequent acetylation, l,2-di-O-acetyl-3-O-benzyl-5-deoxy-5-ethoxycarbonylmethyl-D-ribofuranose was obtained, which reacted with persilylated nucleic acid bases to form 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides.

[Oligonucleotide analogues containing internucleotide C3'-CH2-C(O)-NH-C5' bonds].

A dinucleoside bearing an amide internucleotide C3'-CH2-C(O)-NH-C5' bond was synthesized by the interaction of 3'-deoxy-3'-carboxylmethylribothymidine-2',3'-lactone obtained by hydrolysis of 2'-O-acetyl-5'-O-benzoyl-3'-deoxy-3'-ethoxycarboxylmethylribothymidine with 5'-deoxy-5'-amino-3'-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3'-O-(2-cyanoethyl-N,N'-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones.

[Synthesis of 3'-deoxy-3'-carboxymethylnucleosides, precursors of oligonucleotides with an amide internucleoside bond].

An improved method for the synthesis of 3-deoxy-3-carboxymethylnucleosides was suggested. Oxidation of 5-O-benzoyl-l,2-O-isopropylidene-alpha-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo configuration. Acetolysis of the resulting compound with AcOH-Ac2O-CH3SO3H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42-49% from the starting compound.