A novel approach to encode melodies in DNA.

DNA data storage has gained more attention last decades. DNA molecules can be used for encoding of non-biological information and as promising carriers due to greater data capacity, higher duration of the storage, and better technical failures stability. Here we propose a new method for encoding of notes and music in DNA. The encoding technique takes into account the duration and tonality of each note, enabling to encode all seven octaves by assigning a nucleotide sequence to each key. A certain set of short sequences is suggested to define the duration of note sound. The proposed method allows to encode more complicated melodies compared to the approach based on Huffman algorithm.

LAMPrimers iQ: New primer design software for loop-mediated isothermal amplification (LAMP).

Nucleic acids amplification is a widely used technique utilized for different manipulations with DNA and RNA. Although, polymerase chain reaction (PCR) remains the most popular amplification method, isothermal approaches are gained more attention last decades. Among these, loop-mediated isothermal amplification (LAMP) became an excellent alternative to PCR. LAMP requires an increased number of primers and, therefore, is considered a highly specific amplification reaction compared to PCR. LAMP primers design is still a non-trivial task, and all niceties should be taken into account during their selection. Here, we report on a new program called LAMPrimers iQ destined for high-quality LAMP primers design. LAMPrimers iQ is based on an original algorithm considering rigorous criteria for primers selection. Unlike alternative programs, LAMPrimers iQ can process long DNA or RNA sequences, and completely avoid primers that can form homo- and heterodimers. The quality of the primers designed was checked using SARS-CoV-2 coronavirus RNA as a model target. It was shown that primers selected with LAMPrimers iQ provide higher specificity and reliable detection of viral RNA compared to those obtained by alternative programs. The program is available at https://github.com/Restily/LAMPrimers-iQ.

Detection of Specific RNA Targets by Multimerization.

Detection of specific RNA targets via amplification-mediated techniques is widely used in fundamental studies and medicine due to essential role of RNA in transfer of genetic information and development of diseases. Here, we report on an approach for detection of RNA targets based on the particular type of isothermal amplification, namely, reaction of nucleic acid multimerization. The proposed technique requires only a single DNA polymerase possessing reverse transcriptase, DNA-dependent DNA polymerase, and strand-displacement activities. Reaction conditions that lead to efficient detection of the target RNAs through multimerization mechanism were determined. The approach was verified by using genetic material of the SARS-CoV-2 coronavirus as a model viral RNA. Reaction of multimerization allowed to differentiate the SARS-CoV-2 RNA-positive samples from the SARS-CoV-2 negative samples with high reliability. The proposed technique allows detection of RNA even in the samples, which were subjected to multiple freezing-thawing cycles.

Reverse transcriptase-free detection of viral RNA using Hemo Klentaq DNA polymerase.

COVID-19 pandemic highlighted the demand for the fast and reliable detection of viral RNA. Although various methods for RNA amplification and detection have been proposed, some limitations, including those caused by reverse transcription (RT), need to be overcome. Here, we report on the direct detection of specific RNA by conventional polymerase chain reaction (PCR) requiring no prior RT step. It was found that Hemo KlenTaq (HKTaq), which is posed as DNA-dependent DNA polymerase, possesses reverse transcriptase activity and provides reproducible amplification of RNA targets with an efficiency comparable to common RT-PCR. Using nasopharyngeal swab extracts from COVID-19-positive patients, the high reliability of SARS-CoV-2 detection based on HKTaq was demonstrated. The most accurate detection of specific targets are provided by nearby primers, which allow to determine RNA in solutions affected to multiple freeze-thaw cycles. HKTaq can be used for elaboration of simplified amplification techniques intended for the analysis of any specific RNA and requiring only one DNA polymerase.

Encoding of non-biological information for its long-term storage in DNA.

In 2019, at the World Economic Forum, DNA data storage was indicated as one of the breakthroughs expected to radically impact the global socio-economic order. Indeed, dry DNA is a relatively stable substance and an extremely capacious information carrier. One gram of DNA can hold up to 455 exabytes, provided that one nucleotide encodes two bits of information. In this critical review, the main attention is paid to nucleinography, meaning the conversion of digital data into nucleotide sequences. The evolution and diversity of approaches intended for encoding data with nucleotides are demonstrated. The most noticeable examples of storing minor as well as considerable quantities of non-biological information in DNA are given. Some issues of DNA data storage are also reported.

Convective polymerase chain reaction in standard microtubes.

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 µl reaction volume, 2) the supply of external temperature 145°Ð¡ for the denaturation zone and 0°Ð¡ for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.

A new digital approach to SNP encoding for DNA identification.

Identification of individuals has become an urgent problem for mankind. In the last three decades, STR-based DNA identification has actively evolved along with traditional biometric methods. Nonetheless, single-nucleotide polymorphisms (SNPs) are now of great interest and a number of relevant SNP panels have been proposed for DNA identification. Here, a simple approach to SNP data digitization that can provide assigning a unique genetic identification number (GIN) to each person is proposed. The key points of this approach are as follows: 1) SNP data are digitized as whole 4-bit boxes in the most convenient binary format, where character "1" (YES) is assigned to revealed nucleotides, and character "0" (NO) to missing nucleotides after SNP-typing; 2) all SNPs should be considered tetra-allelic. Calculations showed that a 72-plex SNP panel is enough to provide the population with unique GINs, which can be represented in digital (binary or hexadecimal) or graphic (linear or two-dimensional) formats. Simple software for SNP data processing and GINs creation in any format was written. It is likely that the national and global GIN databases will facilitate the solution of problems related to identification of individuals or human biological materials. The proposed approach may be extended to other living organisms as well.

Enhancement of PCR efficiency using mono- and disaccharides.

Polymerase chain reaction is the most commonly used approach for nucleic acids amplification. Despite the variety of PCR methods have been proposed, new techniques are being developed to improve this reaction. We found that, in general, mono- and disaccharides can serve as effective PCR enhancers. Unlike oligo- and polysaccharides, low molecular-weight carbohydrates accelerate amplification and increase products yield. The ability of carbohydrates to enhance PCR is not related to their reducing property. The best result was obtained for sucrose providing the most specific and reliable amplification. The effect of carbohydrates is leveled as the size of the amplification region increases.

Identification of pyrrolo-pyridine derivatives as novel class of antibacterials.

A series of 5-oxo-4H-pyrrolo[3,2-b]pyridine derivatives was identified as novel class of highly potent antibacterial agents during an extensive large-scale high-throughput screening (HTS) program utilizing a unique double-reporter system-pDualrep2. The construction of the reporter system allows us to perform visual inspection of the underlying mechanism of action due to two genes-Katushka2S and RFP-which encode the proteins with different imaging signatures. Antibacterial activity of the compounds was evaluated during the initial HTS round and subsequent rescreen procedure. The most active molecule demonstrated a MIC value of 3.35 µg/mL against E. coli with some signs of translation blockage (low Katushka2S signal) and no SOS response. The compound did not demonstrate cytotoxicity in standard cell viability assay. Subsequent structural morphing and follow-up synthesis may result in novel compounds with a meaningful antibacterial potency which can be reasonably regarded as an attractive starting point for further in vivo investigation and optimization.

Substituted Furanocoumarins as Novel Class of Antibacterial Translation Inhibitors.

INTRODUCTION: A variety of organic compounds has been reported to have antibacterial activity. However, antimicrobial resistance is one of the main problems of current anti-infective therapy, and the development of novel antibacterials is one of the main challenges of current drug discovery. METHODS: Using our previously developed dual-reporter High-Throughput Screening (HTS) platform, we identified a series of furanocoumarins as having high antibacterial activity. The construction of the reporter system allows us to differentiate three mechanisms of action for the active compounds: inhibition of protein synthesis (induction of Katushka2S), DNA damaging (induction of RFP) or other (inhibition of bacterial growth without reporter induction). RESULTS: Two primary hit-molecules of furanocoumarin series demonstrated relatively low MIC values comparable to that observed for Erythromycin (Ery) against E. coli and weakly induced both reporters. Dose-dependent translation inhibition was shown using in vitro luciferase assay, however it was not confirmed using C14-test. A series of close structure analogs of the identified hits was obtained and investigated using the same screening platform. Compound 19 was found to have slightly lower MIC value (15.18 µM) and higher induction of Katushka2S reporter in contrast to the parent structures. Moreover, translation blockage was clearly identified using both in vitro luciferase assay and C14 test. The standard cytotoxicity test revealed a relatively low cytotoxicity of the most active molecules. CONCLUSION: High antibacterial activity in combination with low cytotoxicity was demonstrated for a series of furanocoumarins. Further optimization of the described structures may result in novel and attractive lead compounds with promising antibacterial efficiency.

Identification of Novel Antibacterials Using Machine Learning Techniques.

Many pharmaceutical companies are avoiding the development of novel antibacterials due to a range of rational reasons and the high risk of failure. However, there is an urgent need for novel antibiotics especially against resistant bacterial strains. Available in silico models suffer from many drawbacks and, therefore, are not applicable for scoring novel molecules with high structural diversity by their antibacterial potency. Considering this, the overall aim of this study was to develop an efficient in silico model able to find compounds that have plenty of chances to exhibit antibacterial activity. Based on a proprietary screening campaign, we have accumulated a representative dataset of more than 140,000 molecules with antibacterial activity against Escherichia coli assessed in the same assay and under the same conditions. This intriguing set has no analogue in the scientific literature. We applied six in silico techniques to mine these data. For external validation, we used 5,000 compounds with low similarity towards training samples. The antibacterial activity of the selected molecules against E. coli was assessed using a comprehensive biological study. Kohonen-based nonlinear mapping was used for the first time and provided the best predictive power (av. 75.5%). Several compounds showed an outstanding antibacterial potency and were identified as translation machinery inhibitors in vitro and in vivo. For the best compounds, MIC and CC50 values were determined to allow us to estimate a selectivity index (SI). Many active compounds have a robust IP position.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform.

AIM AND OBJECTIVE: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. MATERIALS AND METHODS: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. RESULTS: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. CONCLUSION: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.