A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope

Анализ эффективности очистки HEX-олигонуклеотидов, полученных с использованием модифицированного метода деблокирования

A new way of deblocking of synthetic hexachlorofluorescein-oligodeoxyribonucleotides (HEX-ODN) prevents the modification of the label and the accumulation of difficult to separated by-product. It was shown that this method increases the efficiency of purification HEX-ON and the sensitivity of DNA diagnostics.Новый способ деблокирования синтетических гексахлорфлуоресцеин-олигодезоксирибонук-леотидов (HEX-ODN) препятствует модификации метки и накоплению трудно отделяемого побочного продукта. Показано, что его применение увеличивает эффективность очистки HEX-ODN и чувствительность ДНК-диагностики

Оптимизация твердофазного синтеза полиамидных миметиков нуклеиновых кислот с применением MALDI-TOF-МАСС-спектрометрии

The solid-phase synthesis of a tetrameric model sequence of polyamide nucleic acid mimetics (PANAM) incorporating terminal chiral and charged unit was performed. As a result during the synthesis and cleavage from the resin the tendency of PANAM with the N-terminal negatively charged monomer to cyclization with simultaneous N-acyl transfer of carboxyethyl base residue was found. Application of MALDI-TOF mass - spectrometry allowed us to find suitable conditions for obtaining PANAM oligomers without the significant amount of by-products.Осуществлен твердофазный синтез тетрамерной модельной последовательности поли-амидных миметиков нуклеиновых кислот (ПАНКМ) с включением концевого хирального заряженного звена. Выявлена склонность ПАНКМ, содержащих N -концевой отрицательно заряженный мономер на основе L -глутаминовой кислоты, к циклизации с одновременным N -ацильным переносом карбоксиэтильного производного нуклеинового основания в ходе синтеза и отщепления со смолы. Использование MALDI - TOF -масс-спектрометрии в ходе эксперимента позволило найти подходящие условия отщепления олигомеров ПАНКМ, приводящие к существенному снижению количества побочных продуктов

Genetic engineering of peptide hormones : III. Cloning of cDNA of porcine growth hormone and construction of gene for expression of hormone in bacteria

Results are presented of cloning cDNA of procine growth hormone, analysis of its primary structure, and creation of a construction capable of expression of this cDNA in Esqheriahia coti cells. It is shown that in the population of mRNA coding porcine growth hormone, heterogeneity is noted which is manifested not only at the level of the nucleotide sequence, but also is reflected in the amino acid sequence of the mature hormone

Analysis of efficiency of purification of HEX-oligonucleotides obtained by modified deblocking

A new way of deblocking of synthetic hexachlorofluorescein-oligodeoxyribonucleotides (HEX-ODN) prevents the modification of the label and the accumulation of difficult to separated by-product. It was shown that this method increases the efficiency of purification HEX-ON and the sensitivity of DNA diagnostics

OPTIMIZATION OF SOLID-PHASE SYNTHESIS OF POLYAMIDE NUCLEIC ACID MIMETICS WITH APPLICATION OF MALDI-TOF MASS-SPECTROMETRY

The solid-phase synthesis of a tetrameric model sequence of polyamide nucleic acid mimetics (PANAM) incorporating terminal chiral and charged unit was performed. As a result during the synthesis and cleavage from the resin the tendency of PANAM with the N-terminal negatively charged monomer to cyclization with simultaneous N-acyl transfer of carboxyethyl base residue was found. Application of MALDI-TOF mass - spectrometry allowed us to find suitable conditions for obtaining PANAM oligomers without the significant amount of by-products

A Solution to the Common Problem of the Synthesis and Applications of Hexachlorofluorescein Labeled Oligonucleotides.

A common problem of the preparation of hexachlorofluorescein labeled oligonucleotides is the transformation of the fluorophore to an arylacridine derivative under standard ammonolysis conditions. We show here that the arylacridine byproduct with distinct optical characteristics cannot be efficiently separated from the major product by HPLC or electrophoretic methods, which hampers precise physicochemical experiments with the labeled oligonucleotides. Studies of the transformation mechanism allowed us to select optimal conditions for avoiding the side reaction. The novel method for the post-synthetic deblocking of hexachlorofluorescein-labeled oligodeoxyribonucleotides described in this paper prevents the formation of the arylacridine derivative, enhances the yield of target oligomers, and allows them to be proper real-time PCR probes

Parallel-stranded DNA with mixed AT/GC composition: role of trans G·C base pairs in sequence dependent helical stability

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions

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