CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids.

An enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.

With the advent of CRISPR-Cas9 gene editing, we now have to hand a simple two-component system amendable to silencing and knock-in editing effectively any gene. Yet we must not forget that the implications of immunotoxicity along with the poor stability and specificity of canonical nucleic acids hold enormous challenges for in vivo applications, especially in gene therapy. Our study endorses the feasibility of the enzymatic approach to incorporate nucleobase modifications into the CRISPR-Cas9 system unveiling the tolerance of Cas9 to N1-methylpseudouridine (m1Ψ)- and emissive thienoguanosine (thG)-modified sgRNA as well as thus far uncharted structural requirements for ensuring proper PAM recognition.

Conformational Morphing by a DNA Analogue Featuring 7-Deazapurines and 5-Halogenpyrimidines and the Origins of Adenine-Tract Geometry.

Several efforts are currently directed at the creation and cellular implementation of alternative genetic systems composed of pairing components that are orthogonal to the natural dA/dT and dG/dC base pairs. In an alternative approach, Watson-Crick-type pairing is conserved, but one or all of the four letters of the A, C, G, and T alphabet are substituted by modified components. Thus, all four nucleobases were altered to create halogenated deazanucleic acid (DZA): dA was replaced by 7-deaza-2'-deoxyadenosine (dzA), dG by 7-deaza-2'-deoxyguanosine (dzG), dC by 5-fluoro-2'-deoxycytidine (FdC), and dT by 5-chloro-2'-deoxyuridine (CldU). This base-pairing system was previously shown to retain function in Escherichia coli. Here, we analyze the stability, hydration, structure, and dynamics of a DZA Dickerson-Drew Dodecamer (DDD) of sequence 5'-FdC-dzG-FdC-dzG-dzA-dzA-CldU-CldU-FdC-dzG-FdC-dzG-3'. Contrary to similar stabilities of DDD and DZA-DDD, osmotic stressing revealed a dramatic loss of hydration for the DZA-DDD relative to that for the DDD. The parent DDD 5'-d(CGCGAATTCGCG)-3' features an A-tract, a run of adenosines uninterrupted by a TpA step, and exhibits a hallmark narrow minor groove. Crystal structures─in the presence of RNase H─and MD simulations show increased conformational plasticity ("morphing") of DZA-DDD relative to that of the DDD. The narrow dzA-tract minor groove in one structure widens to resemble that in canonical B-DNA in a second structure. These changes reflect an indirect consequence of altered DZA major groove electrostatics (less negatively polarized compared to that in DNA) and hydration (reduced compared to that in DNA). Therefore, chemical modifications outside the minor groove that lead to collapse of major groove electrostatics and hydration can modulate A-tract geometry.

The Role of Tyr-His-Trp Triad and Water Molecule Near the N1-Atom of 2-Hydroperoxycoelenterazine in Bioluminescence of Hydromedusan Photoproteins: Structural and Mutagenesis Study.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the "catalytic function" by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins.

In Vivo Assembly and Expression of DNA Containing Non-canonical Bases in the Yeast Saccharomyces cerevisiae.

Chemically modified nucleic acids are of utmost interest in synthetic biology for creating a regulable and sophisticated synthetic system with tailor-made properties. Implanting chemically modified nucleic acids in microorganisms might serve biotechnological applications, while using them in human cells might lead to new advanced medicines. Previously, we reported that a fully modified DNA sequence (called DZA) composed of the four base-modified nucleotides - 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine and 5-fluorocytosine - could function as a genetic template in prokaryotic cells, Escherichia coli. Here, we report the synthesis of long, partially, or fully modified DZA fragments that encode the yeast-enhanced red fluorescent protein (yEmRFP). The DZA sequences were directly introduced in the genome of the eukaryotic cells, Saccharomyces cerevisiae, via the yeast natural homologous recombination machinery. The simple and straightforward DZA cloning strategy reported here might be of interest to scientists working in the field of xenobiology in yeast.

Highly stable hexitol based XNA aptamers targeting the vascular endothelial growth factor.

Biomedical applications of nucleic acid aptamers are limited by their rapid degradation in biological fluids and generally demand tedious post-selection modifications that might compromise binding. One possible solution to warrant biostability is to directly evolve chemically modified aptamers from xenobiotic nucleic acids (XNAs). We have isolated fully modified 2'-O-methyl-ribose-1,5-anhydrohexitol nucleic acid (MeORNA-HNA) aptamers targeting the rat vascular endothelial growth factor 164 (rVEGF164). Three sequences have been identified that interact with the target protein with affinities in the low-nanomolar range and HNA modifications appeared to be mandatory for their tight binding. The evolution of these XNA aptamers was accomplished using an in vitro selection procedure starting from a fully sugar-modified library containing a 20mer 2'-OMe-ribonucleotide region followed by a 47mer HNA sequence. The high binding affinity and selectivity of the selected aptamers were confirmed by several methods including gel-shift, fluorescence polarisation, and enzyme-linked oligonucleotide assays. The isolated HNA ligands exhibited higher specificity to the rVEGF164 and human VEGF165 isoforms compared to rat VEGF120, while very low binding efficiencies were observed to streptavidin and thrombin. Furthermore, it was clearly demonstrated that the resulting aptamers possessed a superior stability to degradation in human serum and DNase I solutions.

The Network of Replication, Transcription, and Reverse Transcription of a Synthetic Genetic Cassette.

Synthetic nucleic acids, with four non-canonical nucleobases, can function as genetic materials. A comprehensive analysis of PCR amplification, transcription, reverse transcription, and cloning was done to screen for alternative genetic monomers. A small library of six modified nucleobases was selected: the modified 2'-deoxyribonucleoside (dZTPs) and ribonucleoside (rZTPs) triphosphates of 7-deaza-adenine, 5-chlorouracil, 7-deaza-guanine or inosine together with 5-fluorocytosine or 5-bromocytosine. The fragments composed of one to four modified nucleotides (denoted as DZA) have been successfully recognized and transcribed to natural or modified RNA (denoted as RZA) by T7 RNA polymerase. The fully modified RZA fragment could be reverse transcribed and then amplified in the presence of various dZTPs. Noticeably, modified fragments could function as genetic templates in vivo by encoding the 678 base pair gene of a fluorescent protein in bacteria. These results demonstrate the existence of a fully simulated genetic circuit that uses synthetic materials.

In Vivo Expression of Genetic Information from Phosphoramidate-DNA.

Chemically modified genes and genomes with customized properties will become a valuable tool in numerous fields, including synthetic biology, biotechnology, and medicine. These genetic materials are meant to store and exchange information with DNA and RNA while tuning their functionality. Herein, we outline the development of an alternative genetic system carrying phosphoramidate linkages that successfully propagates genetic information in bacteria and at the same time is labile to acidic conditions. The P3'→N5' phosphoramidate-containing DNA (PN-DNA) was enzymatically synthesized by using 5'-amino-2',5'-deoxycytidine 5'-N-triphosphates (NH-dCTPs) as substrates for DNA polymerases and employed to encode antibiotic resistance in Escherichia coli. The resulting PN-DNA can be efficiently destroyed by mild acidic conditions, whereas an unmodified counterpart remains intact. A cloning strategy was proposed for assembling modified fragments into a genome. This method can be of interest to scientists working in the field of orthogonal nucleic acid genes and genomes.

Structure-Activity Relationship Study of a Potent α-Thrombin Binding Aptamer Incorporating Hexitol Nucleotides.

The replacement of one or more nucleotide residues in the potent α-thrombin-binding aptamer NU172 with hexitol-based nucleotides has been devised to study the effect of these substitutions on the physicochemical and functional properties of the anticoagulant agent. The incorporation of single hexitol nucleotides at the T9 and G18 positions of NU172 substantially retained the physicochemical features of the parent oligonucleotide, as a result of the biomimetic properties of the hexitol backbone. Importantly, the NU172-TH 9 mutant exhibited a higher binding affinity toward human α-thrombin than the native aptamer and an improved stability even after 24 h in 90 % human serum, with a significant increase in the estimated half-life. The anticoagulant activity of the modified oligonucleotide was also found to be slightly preferable to NU172. Overall, these results confirm the potential of hexitol nucleotides as biomimetic agents, while laying the foundations for the development of NU172-inspired α-thrombin-binding aptamers.

The interaction of C-terminal Tyr208 and Tyr13 of the first α-helix ensures a closed conformation of ctenophore photoprotein berovin.

Light-sensitive Ca2+-regulated photoprotein berovin is responsible for the bioluminescence of the ctenophore Beroe abyssicola. It shares many properties of hydromedusan photoproteins although the degree of identity of its amino acid sequence with those of photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan photoproteins that supports a closed conformation of the internal cavity of the photoprotein molecule with bound 2-hydroperoxycoelenterazine. The C- and N-terminal hydrogen bond network is necessary to properly isolate the photoprotein active site from the solvent and consequently to provide a high quantum yield of the bioluminescence reaction. In order to find out which berovin residues perform the same function we modified the N- and C-termini of the protein by replacing or deleting various amino acid residues. The studies on berovin mutants showed that the interaction between C-terminal Tyr208 and Tyr13 localized in the first α-helix of the photoprotein is important for the stabilization and proper orientation of the oxygenated coelenterazine adduct within the internal cavity as well as for supporting the closed photoprotein conformation. We also suggest that the interplay between Tyr residues in ctenophore photoproteins occurs rather through the π-π interaction of their phenyl rings than through hydrogen bonds as in hydromedusan photoproteins.

Bioluminescent Properties of Semi-Synthetic Obelin and Aequorin Activated by Coelenterazine Analogues with Modifications of C-2, C-6, and C-8 Substituents.

Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated coelenterazine, 2-hydroperoxycoelenterazine, is tightly bound. Alongside with native coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel coelenterazine analogues and the recently reported coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain coelenterazine analogues.

EXPRESSION OF THE RENIN-ANGIOTENSIN SYSTEM COMPONENTS IN ONCOLOGIC DISEASES.

The literature devoted to changes in the expression of the renin-angiotensin system (RAS) proteins of cancer cells was analyzed. The dynamics of RAS protein expression in malignant tumors and the possible role of epigenetic mechanisms in these processes are briefly reviewed. Through research of the epigenetic mechanisms in cancer, principally new techniques for their correction based on the use of selective regulatory systems of covalent modification of histone proteins (for example, deacetylase inhibitor) and microRNA synthesis technologies have been developed. Literature data show promising pharmacological correction of epigenetic modification of chromatin in the treatment of cancer.

Base-Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology.

The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.

Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca(2+)-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction.

Chemical Morphing of DNA Containing Four Noncanonical Bases.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo.

Structures of the Ca2+-regulated photoprotein obelin Y138F mutant before and after bioluminescence support the catalytic function of a water molecule in the reaction.

Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F obelin mutant before and after bioluminescence are reported at 1.72 and 1.30 Šresolution, respectively. The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild-type obelin gives clear evidence that the substitution of Tyr by Phe does not affect the overall structure of both Y138F obelin and its product following Ca(2+) discharge compared with the corresponding conformational states of wild-type obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild-type obelins, there is a substantial difference: in the cavity of Y138F obelin a water molecule corresponding to W2 in wild-type obelin is not found. However, in Ca(2+)-discharged Y138F obelin this water molecule now appears in the same location. This finding, together with the observed much slower kinetics of Y138F obelin, clearly supports the hypothesis that the function of a water molecule in this location is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion before its decomposition into the excited-state product. Although obelin differs from other hydromedusan Ca(2+)-regulated photoproteins in some of its properties, they are believed to share a common mechanism.

Hydrogen-bond networks between the C-terminus and Arg from the first α-helix stabilize photoprotein molecules.

Previous studies have stated that aequorin loses most of its bioluminescence activity upon modification of the C-terminus, thus limiting the production of photoprotein fusion proteins at its N-terminus. In the present work, we investigate the importance of the C-terminal proline and the hydrogen bonds it forms for photoprotein active complex formation, stability and functional activity. According to the crystal structures of obelin and aequorin, two Ca(2+)-regulated photoproteins, the carboxyl group of the C-terminal Pro forms two hydrogen bonds with the side chain of Arg21 (Arg15 in aequorin case) situated in the first α-helix. Whereas, deletion or substitution of the C-terminal proline could noticeably change the bioluminescence activity, stability or the yield of an active photoprotein complex. Therefore, modifications of the first α-helix Arg has a clear destructive effect on the main photoprotein properties. A C-terminal hydrogen-bond network is proposed to be important for the stability of photoprotein molecules towards external disturbances, when taking part in the formation of locked protein conformations and isolation of coelenterazine-binding cavities.

Primary production in the bays of the novaya zemlya archipelago (Kara Sea) in the contrasting glacial and non-glacial environmental conditions.

Ongoing warming is leading to the accelerated shrinkage of glaciers located on Arctic islands. Consequently, the influence of glacial meltwater on phytoplankton primary production in Arctic bays becomes critically important in an era of warming. This work studies the spatiotemporal variation of primary production and chlorophyll a concentration in the bays along the eastern coast of the Novaya Zemlya archipelago. Data were collected during nine cruises performed from July to October (2013-2022). The effect of underwater photosynthetically available radiation (PAR) and nutrients on primary production was assessed separately for bays influenced by glacial meltwater (glacial bays) and those without such influence (non-glacial bays). The median value of water column-integrated primary production (IPP) for all bays was 38 mgC m-2 d-1, characterizing them as oligotrophic areas. IPP in non-glacial bays was found to be 2.3-fold and 1.4-fold higher than that in glacial bays during summer and autumn, respectively. Underwater PAR was the main abiotic factor determining IPP during the ice-free period. In the entire bays nutrient concentrations were high, exceeding the limiting values for growth and photosynthesis of phytoplankton. It was concluded that the high turbidity from glacial meltwater runoff leads to decreased underwater PAR and, consequently, to a decline in IPP. This study demonstrates that rapid warming could have a negative impact on the productivity of high Arctic bays and their adjacent areas.

Oxygen activation of apo-obelin-coelenterazine complex.

Ca(2+) -regulated photoproteins use a noncovalently bound 2-hydroperoxycoelenterazine ligand to emit light in response to Ca(2+) binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo-obelin-coelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7-protonated and C2(-) anionic forms, and that oxygen shifts the equilibrium in favor of the C2(-) anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2-hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca(2+) -triggering of the bioluminescence reaction.

Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.

Ca(2+)-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor-acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His-Trp-Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His-Trp-Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.

Intracavity enhancement of GFP fluorescence induced by femtosecond laser pulses.

The phenomenon of fluorescence is widely used in molecular biology for studying the interaction of light with biological objects. In this article, we present an experimental investigation of the enhancement of laser-induced fluorescence of Clytia gregaria green fluorescent protein. The laser-induced fluorescence method applied in our work combines the advantages of femtosecond laser pulses and a photonic crystal cavity, with the time dependence of the fluorescence signal studied. It is shown that a green fluorescent protein solution placed in a microcavity and excited by femtosecond laser pulses leads to an increase in fluorescence on the microcavity modes, which can be estimated by two orders of magnitude. The dependences of fluorescence signal saturation on the average integrated optical pump power are demonstrated and analyzed. The results obtained are of interest for the development of potential applications of biophotonics and extension of convenient methods of laser-induced fluorescence.