Biomedical subjects

Dmitry M Kolpashchikov

Publications and source records attributed to Dmitry M Kolpashchikov.

8 recordsLinked to original sources

PubMed2026

Tripled-Stranded Antisense Oligonucleotide for Biomarker-Activated Suppression of Essential Genes.

Conditional activation of antisense oligonucleotides (ASOs) is a promising strategy for selective suppression of cancer cells without affecting normal cells. In this study, we developed a tripled-stranded ASO (tsASO) that is rendered inactive through complexation with two additional oligonucleotides. The key innovation is the use of partial overlap between the parent ASO and the biomarker sequence, combined with toehold-mediated strand displacement, enabling precise conditional activation. The tsASO effectively triggered RNase H-mediated degradation of DYNC1I2 and DARS1 RNAs exclusively in the presence of the ERBB2 sequence. In cell-free systems, the tsASO demonstrated high cleavage efficiency (up to 81%), comparable to the parent ASO efficiency, with minimal background activity in the absence of the biomarker sequence, validating the concept at the molecular level. However, in cells using lipid-based transfection, the tsASO exhibited nonspecific cytotoxicity that did not correlate with biomarker presence or target gene expression. Detailed analysis showed no clear support for known sequence-driven toxicity mechanisms (CpG/TLR9, G-quadruplexes) in the nonimmune cell lines, suggesting that the primary limitation is intracellular delivery rather than the tsASO design. Future work should focus on optimizing delivery platforms to achieve controlled cellular uptake and biomarker-dependent release, unlocking the therapeutic potential of this conditional gene silencing approach.

Oligonucleotides, Antisense↗

PubMed2006

A binary DNA probe for highly specific nucleic Acid recognition.

A new concept for nucleic acid probe design is reported. The extremely high selectivity of the probe is predetermined by cooperative hybridization of the two relatively short (10 nucleotide) DNA hairpin fragments to the analyte. A binary DNA probe fluorescently reports the presence of 0.5% of the analyte in excess amount of a single base substituted oligodeoxyribonucleotide and distinguishes single nucleotide substitutions at any position of a 20-mer oligonucleotide at room temperature.

Base Sequence↗

PubMed2005

Binary malachite green aptamer for fluorescent detection of nucleic acids.

A new probe that can fluorescently report the presence of specific nucleic acids in solution with extremely high selectivity was developed. The probe consists of malachite green-a triphenylmethane dye-and two short RNA strands, each of which comprises a fragment complementary to an analyte molecule and a fragment of a malachite green aptamer (MGA). The two RNA strands form MGA upon hybridization to the adjacent positions of the nucleic acid analyte. MGA is able to bind malachite green and enhance the fluorescence of the dye, thus monitoring the presence of the nucleic acid in solution. The probe reliably discriminates against 41 out of 42 possible single nucleotide substitutions in 14-mer DNA analyte at room temperature in physiological buffer. Consisting of unmodified RNA strands, which can be expressed in living cells, binary MGA probe represents a promising instrument for real-time nucleic acid monitoring in vivo.

Microscopy, Fluorescence↗

PubMed2005

Boolean control of aptamer binding states.

Molecules that perform Boolean calculations can analyze a series of inputs and make a decision to produce or not to produce an output, based on the presence or absence of inputs and a particular formula they encode. We now report that molecular computation performed by deoxyribozyme-based logic gates can be used to control the functional state of aptamers, switching them on or off based on the outcome of such computation.

Computers, Molecular↗

PubMed2004

Modular aptameric sensors.

We report the first examples of modular aptameric sensors, which transduce recognition events into fluorescence changes through allosteric regulation of noncovalent interactions with a fluorophore. These sensors consist of: (a) a reporting domain, which signals the binding event of an analyte through binding to a fluorophore; (b) a recognition domain, which binds the analyte; and (c) a communication module, which serves as a conduit between recognition and signaling domains. We tested recognition regions specific for ATP, FMN, and theophylline in combinations with malachite green binding aptamer as a signaling domain. In each case, we were able to obtain a functional sensor capable of responding to an increase in analyte concentration with an increase in fluorescence. Similar constructs that consist only of natural RNA could be expressed in cells and used as sensors for intracellular imaging.

Adenosine Triphosphate↗

PubMed2004

Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1 subunit.

Influenza virus RNA polymerase is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the viral RNA genome. The catalytic site for RNA polymerization is located on the PB1 subunit. To identify the primer ATP-binding site, we have employed a highly selective cross-linking technique: three structurally diverse ATP analogues with reactive groups on their phosphate moieties were first cross-linked to the viral RNA polymerase, and the cross-linked nucleotides were then elongated to dinucleotides by adding the second substrate [alpha-(32)P]GTP. Only the ATP analogues tethered to the primer-binding site could be elongated to radioactive AG dinucleotides. Using this catalytically competent cross-linking procedure, the PB1 subunit was found to be the only labeled subunit. Limited proteolysis of the labeled PB1 by V8 protease revealed the segment between amino acids 179 and 297 as the only bearer of the radioactive label. Thus, we concluded that this region of PB1 faces the 5' end of the primer nucleotide. In support of this prediction, the cross-linked dinucleotides were further elongated up to 8-10 nucleotides in length upon addition of all four substrates. This result suggests that the substantial mass of RNA can be accommodated between the binding site for the primer (and nascent RNA) and the catalytic center of RNA polymerization.

Binding Sites↗

PubMed2003

Superselective labelling of proteins: approaches and techniques.

Affinity labelling is a popular method used for the study of macromolecules and their interactions with ligands. The method is based on the targeted delivery of a chemically cross-linkable group, attached to a reactive molecule with affinity for a particular site in the biopolymer of interest. In complex multicomponent systems, the applications of affinity labelling are restricted by the tendency of the reagents to randomly label nontargetted molecules. This review highlights techniques developed to minimize non-specific cross-linking and to achieve high selectivity for the labelling of target protein. Such techniques might be termed 'superselective labelling', as opposed to traditional, less selective approaches.

Animals↗

PubMed2002

Binary system for selective photoaffinity labeling of base excision repair DNA polymerases.

A system of photoaffinity reagents for selective labeling of DNA polymerases in extracts has been examined. To create the photoreactive DNA probe in situ, DNA substrates containing a synthetic abasic site are incubated in mouse embryonic fibroblast (MEF) cellular extract in the presence of base-substituted arylazido derivatives of dNTPs. This results in synthesis of a photoreactive long patch base excision repair (BER) intermediate. The arylazido photoreactive group is then activated through energy transfer from the pyrene group of a dNTP analog (Pyr-dUTP), following 365 nm UV light exposure. Pyr-dUTP binds to the active site of DNA polymerases, and the pyrene group, when excited by 365 nm UV light, activates the nearby photoreactive group in the BER intermediate resulting in crosslinking of DNA-bound DNA polymerases. Under these conditions, various DNA binding proteins that are unable to bind Pyr-dUTP are not crosslinked to DNA. DNA polymerase beta is the predominant crosslinked protein observed in the MEF extract. In contrast, several other DNA binding proteins are labeled under conditions of direct UV light activation of the photoreactive group at 312 nm. This study illustrates use of a new method of selective labeling of DNA polymerases in a crude cellular extract.

Animals↗