Imino Proton Research Articles

Optimising in-cell NMR acquisition for nucleic acids.

Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppressionfor in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

Efficient Synthetic Access to Stable Isotope Labelled Pseudouridine Phosphoramidites for RNA NMR Spectroscopy.

Here we report the efficient synthetic access to 13C/15N-labelled pseudouridine phosphoramidites, which were incorporated into a binary H/ACA box guide RNA/product complex comprising 77 nucleotides (nts) in total and into a 75 nt E. coli tRNAGly. The stable isotope (SI) labelled pseudouridines were produced via a highly efficient chemo-enzymatic synthesis. 13C/15N labelled uracils were produced via chemical synthesis and enzymatically converted to pseudouridine 5'-monophosphate (ΨMP) by using YeiN, a Ψ-5'-monophosphate C-glycosidase. Removal of the 5'-phosphate group yielded the desired pseudouridine nucleoside (Ψ), which was transformed into a phosphoramidite building suitable for RNA solid phase synthesis. A Ψ -building block carrying both a 13C and a 15N label was incorporated into a product RNA and the complex formation with a 63 nt H/ACA box RNA could be observed via NMR. Furthermore, the SI labelled pseudouridine building block was used to determine imino proton bulk water exchange rates of a 75 nt E. coli tRNAGly CCmnm5U, identifying the TΨC-loop 5-methyluridine as a modifier of the exchange rates. The efficient synthetic access to SI-labelled Ψ building blocks will allow the solution and solid-state NMR spectroscopic studies of Ψ containing RNAs and will facilitate the mass spectrometric analysis of Ψ-modified nucleic acids.

DYNAMICS OF COMPLEMENTARY BASE-PAIRS OF DREW-DICKERSON DODECAMER ON THE MILLIAND MICROSECOND TIME SCALE ACCORDING TO THE DATA OF NUCLEAR SPIN RELAXATION DISPERSION R1ρ OF IMINO PROTONS

Drew-Dickerson Dodecamer (DDD) is one of the well-known DNA duplexes. It has been sufficiently well studied by various structure determination methods and appears to be a standard for developing new experimental techniques and molecular mechanical force fields. For this reason, the information about the dynamics of DDD on different time scales is important for constructing more accurate structural-dynamic models and carrying out molecular dynamics calculations of long-term time trajectories that allow simulation of real processes. In this work, the information on the dynamics of DDD base-pairs on the milli- and microsecond time scale, currently missing from the literature, was obtained by means of imino protons’ nuclear spin relaxation (R1ρ) dispersion. It is shown for all the base-pairs, except for terminal and pre-terminal ones, that any dynamic processes in the range of R1ρ method are not observed up to 300 K, but proceed on a faster time scale. For the pre-terminal GC pair, a pronounced dependence of the relaxation R1ρ on the applied spin-lock field strength is observed, which allowed us to extract the information about the pair closing dynamics using a fast exchange model with highly skewed state populations. These data supplement the information obtained previously on the dynamics of DDD complementary base-pairs by exchange-based water magnetization transfer experiments and provide a more complete view of the processes observed in DDD in relation to base-pair opening-closing.

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p.

The s2m, a highly conserved 41-nt hairpin structure in the SARS-CoV-2 genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and the subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a >99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR spectroscopy assignments comparing the imino proton resonance region of s2m and the s2m G15U at 19°C, we show that the U15-A29 base pair closes, resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we show that the s2m G15U mutation drastically impacts the binding of host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on immune responses.

Pressure pushes tRNALys3 into excited conformational states

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Dynamics of 8-Oxoguanine in DNA: Decisive Effects of Base Pairing and Nucleotide Context.

8-Oxo-7,8-dihydroguanine (oxoG), an abundant DNA lesion, can mispair with adenine and induce mutations. To prevent this, cells possess DNA repair glycosylases that excise either oxoG from oxoG:C pairs (bacterial Fpg, human OGG1) or A from oxoG:A mispairs (bacterial MutY, human MUTYH). Early lesion recognition steps remain murky and may include enforced base pair opening or capture of a spontaneously opened pair. We adapted the CLEANEX-PM NMR protocol to detect DNA imino proton exchange and analyzed the dynamics of oxoG:C, oxoG:A, and their undamaged counterparts in nucleotide contexts with different stacking energy. Even in a poorly stacking context, the oxoG:C pair did not open easier than G:C, arguing against extrahelical base capture by Fpg/OGG1. On the contrary, oxoG opposite A significantly populated the extrahelical state, which may assist recognition by MutY/MUTYH.

2-Amino purine as a probe of DNA deformability in damaged DNA.

2-Amino purine (2AP) is an isomer of adenine (6-aminopurine) that, unlike the canonical DNA bases, is highly fluorescent when free in solution. The fluorescence quantum yield of 2AP in water is 0.68, and its fluorescence intensity decay is monoexponential with a single lifetime of 10-11 ns. When incorporated into DNA, 2AP fluorescence is quenched strongly by interactions with its neighboring bases. Inter-base interactions in duplex DNA give rise to a multiexponential decay that reflects the highly heterogeneous environment sensed by the probe. We used steady-state and time-resolved 2AP fluorescence to probe DNA dynamics around a lesion. In this work, we focused on uracil, a highly mutagenic and common lesion in DNA. In the cell, uracil is removed by Uracil DNA-glycosylase (UNG). UNG excision efficiency depends on DNA sequence, and this research is part of a broader study aimed to elucidate the underlying principles that dictate UNG substrate specificity. Duplex DNA substrates were prepared by annealing uracil-containing oligonucleotides with complementary strands containing 2AP opposite to the uracil. Fluorescence lifetimes were measured with 40 ps resolution, and results were analyzed together with the measured fluorescence quantum yields to calculate the fraction of ultra-quenched 2AP molecules. A higher fraction indicates a higher fraction of highly stacked (and therefore efficiently quenched) 2AP molecules, which we interpret as evidence for a less deformable substrate. This interpretation is supported by NMR imino proton exchange measurements and molecular dynamics simulations on the same substrates.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

DNA sequence effects have been identified for many glycosylases and alkyl-transferases that repair base mismatches, uracil bases, alkylated bases, etc. Here, we focused on understanding how DNA sequence impacts the repair of uracil, a highly mutagenic and common lesion in DNA. Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes uracil from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence. We show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. We observed a strong correlation between UNG efficiency and substrate flexibility, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. A better understanding of the fundamental principles that dictate UNG activity has broad implications in diverse fields, from cancer to evolution.

Accurate and Cost-Effective NMR Chemical Shift Predictions for Nucleic Acids Using a Molecules-in-Molecules Fragmentation-Based Method.

We have developed, implemented, and assessed an efficient protocol for the prediction of NMR chemical shifts of large nucleic acids using our molecules-in-molecules (MIM) fragment-based quantum chemical approach. To assess the performance of our approach, MIM-NMR calculations are calibrated on a test set of three nucleic acids, where the structure is derived from solution-phase NMR studies. For DNA systems with multiple conformers, the one-layer MIM method with trimer fragments (MIM1trimer) is benchmarked to get the lowest energy structure, with an average error of only 0.80 kcal/mol with respect to unfragmented full molecule calculations. The MIMI-NMRdimer calibration with respect to unfragmented full molecule calculations shows a mean absolute deviation (MAD) of 0.06 and 0.11 ppm, respectively, for 1H and 13C nuclei, but the performance with respect to experimental NMR chemical shifts is comparable to the more expensive MIM1-NMR and MIM2-NMR methods with trimer subsystems. To compare with the experimental chemical shifts, a standard protocol is derived using DNA systems with Protein Data Bank (PDB) IDs 1SY8, 1K2K, and 1KR8. The effect of structural minimizations is employed using a hybrid mechanics/semiempirical approach and used for computations in solution with implicit and explicit-implicit solvation models in our MIM1-NMRdimer methodology. To demonstrate the applicability of our protocol, we tested it on seven nucleic acids, including structures with nonstandard residues, heteroatom substitutions (F and B atoms), and side chain mutations with a size ranging from ∼300 to 1100 atoms. The major improvement for predicted MIM1-NMRdimer calculations is obtained from structural minimizations and implicit solvation effects. A significant improvement with the explicit-implicit solvation model is observed only for two smaller nucleic acid systems (1KR8 and 7NBK), where the expensive first solvation shell is replaced by the microsolvation model, in which a single water molecule is added for each solvent-exposed amino and imino protons, along with the implicit solvation. Overall, our target accuracy of ∼0.2-0.3 ppm for 1H and ∼2-3 ppm for 13C has been achieved for large nucleic acids. The proposed MIM-NMR approach is accurate and cost-effective (linear scaling with system size), and it can aid in the structural assignments of a wide range of complex biomolecules.

Combining Experimental Restraints and RNA 3D Structure Prediction in RNA Nanotechnology.

Precise RNA tertiary structure prediction can aid in the design of RNA nanoparticles. However, most existing RNA tertiary structure prediction methods are limited to small RNAs with relatively simple secondary structures. Large RNA molecules usually have complex secondary structures, including multibranched loops and pseudoknots, allowing for highly flexible RNA geometries and multiple stable states. Various experiments and bioinformatics analyses can often provide information about the distance between atoms (or residues) in RNA, which can be used to guide the prediction of RNA tertiary structure. In this chapter, we will introduce a platform, iFoldNMR, that can incorporate non-exchangeable imino protons resonance data from NMR as restraints for RNA 3D structure prediction. We also introduce an algorithm, DVASS, which optimizes distance restraints for better RNA 3D structure prediction.

Synthesis of isoindole-diimine and its derivatives without ammonia gas bubbling

Isoindole-diimine, which can be obtained by bubbling ammonia gas into a solution of phthalonitrile derivatives in the presence of Na[Formula: see text] ion, is a final and best precursor for phthalocyanine synthesis. In this study, isoindole-diimine and various derivatives have been prepared without bubbling ammonia gas using in situ generated ammonia that was produced from formamide and a strong alkali such as NaOH and NaNH2. A longer reaction time, higher temperature, and larger amount of formamide were required for phthalonitrile derivatives with electron-donating groups, but this method is recommendable in that an NH3 gas cylinder and concomitant gas regulator, which are not inexpensive, are not required. Attempts were made to interpret the IR and absorption spectra of the resultant diimines. The signals due to imino proton or N-H of diimino compounds were experimentally found to appear at around 8.2[Formula: see text]9.5 ppm in 1H-NMR and ca. 3500–3000 cm[Formula: see text] in IR spectra.

Shedding light on the base-pair opening dynamics of nucleic acids in living human cells

Base-pair opening is a fundamental property of nucleic acids that plays important roles in biological functions. However, studying the base-pair opening dynamics inside living cells has remained challenging. Here, to determine the base-pair opening kinetics inside living human cells, the exchange rate constant ({k}_{{{{{{rm{ex}}}}}}}) of the imino proton with the proton of solvent water involved in hairpin and G-quadruplex (GQ) structures is determined by the in-cell NMR technique. It is deduced on determination of {k}_{{{{{{rm{ex}}}}}}} values that at least some G-C base pairs of the hairpin structure and all G-G base-pairs of the GQ structure open more frequently in living human cells than in vitro. It is suggested that interactions with endogenous proteins could be responsible for the increase in frequency of base-pair opening. Our studies demonstrate a difference in dynamics of nucleic acids between in-cell and in vitro conditions.

Salt Dependence of DNA Binding Activity of Human Transcription Factor Dlx3.

Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5′-TAATT-3′) of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.

HnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression.

Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (TM = 61.2 °C) and G25T (TM = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5′ and 3′ tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.

Rapid assessment of Watson-Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino 1H CEST.

In duplex DNA, Watson-Crick A-T and G-C base pairs (bp's) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for identifying potential Hoogsteen hot spots and for understanding the potential roles of Hoogsteen base pairs in DNA recognition and repair. However, such studies are hampered by the need to prepare C or N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) H CEST experiments employing high-power radiofrequency fields ( 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Watson-Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino H CEST were in very good agreement with counterparts measured using off-resonance C N spin relaxation in the rotating frame (). It is shown that H-H NOE effects which typically introduce artifacts in H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short ( 100 ms). The H CEST experiment can be performed with 10 higher throughput and 100 lower cost relative to C N and enabled Hoogsteen chemical exchange measurements undetectable by . The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization.

Impact of 3-deazapurine nucleobases on RNA properties.

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.

3D Heteronuclear Magnetization Transfers for the Establishment of Secondary Structures in SARS-CoV-2-Derived RNAs.

Multidimensional NOESY experiments targeting correlations between exchangeable imino and amino protons provide valuable information about base pairing in nucleic acids. It has been recently shown that the sensitivity of homonuclear correlations involving RNA’s labile imino protons can be significantly enhanced, by exploiting the repolarization brought about by solvent exchanges. Homonuclear correlations, however, are of limited spectral resolution, and usually incapable of tackling relatively large homopolymers with repeating structures like RNAs. This study presents a heteronuclear-resolved version of those NOESY experiments, in which magnetization transfers between the aqueous solvent and the nucleic acid protons are controlled by selecting specific chemical shift combinations of a coupled 1H–15N spin pair. This selective control effectively leads to a pseudo-3D version of HSQC-NOESY, but with cross-peaks enhanced by ∼2–5× as compared with conventional 2D NOESY counterparts. The enhanced signal sensitivity as well as access to both 15N–1H and 1H–1H NOESY dimensions can greatly facilitate RNA assignments and secondary structure determinations, as demonstrated here with the analysis of genome fragments derived from the SARS-CoV-2 virus.

Selective, pH sensitive, “turn on” fluorescence sensing of carbonate ions by a benzimidazole

Anions play crucial roles in the sustenance of life on earth in many ways. Selective detection of specific anions is important in developing new diagnostic tools and therapeutics. A pH-sensitive & selective benzimidazole-based fluorescent sensor has been developed for rapid detection of carbonate ions which can detect carbonate ions in low nanomolar concentrations. NMR based experiments indicate direct interaction of benzimidazole imino protons with the carbonate ions leading to 1:1 ligand carbonate ion complexation events. This is one of the first reports of benzimidazole sensing carbonate ions with high selectivity which may have implications in disease prevention and toxicity assessment.

The model structure of the hammerhead ribozyme formed by RNAs of reciprocal chirality.

RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA’s resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyse RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the 3D structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson–Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.