Percutaneous distal chevron osteotomy is associated with lower immediate postoperative pain and a greater range of motion than the open technique: A prospective randomized study.

PURPOSE: The results of past studies comparing percutaneous techniques with traditional open techniques for hallux valgus are controversial. Therefore, this study aimed to compare the radiologic and clinical outcomes of percutaneous and open distal chevron osteotomies. METHODS: Seventy-one patients with mild to severe hallux valgus deformity were randomized to undergo percutaneous distal chevron osteotomy (percutaneous group, n = 36) or open distal chevron osteotomy (open group, n = 35) between October 2019 and September 2020. Radiological and clinical outcomes were assessed preoperatively and postoperatively. Outcome measures included the foot and ankle outcome score, foot functional index, visual analogue scale (VAS) scores for pain, range of motion (ROM) of the first metatarsophalangeal (MTP) joint, hallux valgus angle, intermetatarsal angle, and first metatarsal shortening. Additionally, the first metatarsal declination angle was measured to evaluate sagittal malunion. RESULTS: The mean first metatarsal declination angle decreased significantly at 12 months postoperatively in both groups (p = 0.021 and p < 0.001 in the percutaneous and open groups, respectively), and the decrement was significantly greater in the open group (p = 0.033). The mean VAS score for pain on postoperative day one was 4.2 ± 1.9 and 5.3 ± 1.7 in the percutaneous and open groups, respectively (p = 0.019). The mean ROM of the first MTP joint did not change significantly after surgery, from 72.5 ± 7.5 preoperatively to 71.0 ± 9.5 at 12 months postoperatively in the percutaneous group (p = 0.215); however, it decreased significantly from 70.6 ± 7.3 preoperatively to 63.4 ± 10.4 at 12 months postoperatively in the open group (p < 0.001). There were no significant differences between the groups regarding other clinical outcomes. CONCLUSION: The percutaneous group showed a lower immediate pain level at postoperative day 1 and better ROM of the first MTP joint at 12 months postoperatively.

LIN28A loss of function is associated with Parkinson's disease pathogenesis.

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.

Effects of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, cellular uptake, and gene expression.

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.

Longterm Outcomes of Patients Undergoing Liver Transplantation for Acute-on-Chronic Liver Failure.

Recent data have demonstrated >80% 1-year survival probability after liver transplantation (LT) for patients with severe acute-on-chronic liver failure (ACLF). However, longterm outcomes and complications are still unknown for this population. Our aim was to compare longterm patient and graft survival among patients transplanted across all grades of ACLF. We analyzed the United Network for Organ Sharing database for the years 2004-2017. Patients with ACLF were identified using the European Association for the Study of the Liver-Chronic Liver Failure criteria. Kaplan-Meier and Cox regression methods were used to determine patient and graft survival and associated predictors of mortality in adjusted models. A total of 56,801 patients underwent transplantation of which 31,024 (54.6%) had no ACLF, 8757 (15.4%) had ACLF grade 1, 9039 (15.9%) had ACLF grade 2, and 7891 (14.1%) had ACLF grade 3. The 5-year patient survival after LT was lower in the ACLF grade 3 patients compared with the other groups (67.7%; P < 0.001), although after year 1, the percentage decrease in survival was similar among all groups. Infection was the primary cause of death among all patient groups in the first year. Infection was the primary cause of death among all patient groups in the first year. After the first year, infection was the main cause of death in patients transplanted with ACLF grade 1 (32.1%), ACLF grade 2 (33.9%), and ACLF grade 3 (37.6%), whereas malignancy was the predominant cause of death in those transplanted with no ACLF (28.5%). In conclusion, patients transplanted with ACLF grade 3 had lower 5-year survival as compared with patients with ACLF grades 0-2, but mortality rates were not significantly different after the first year following LT. Graft survival was excellent across all ACLF groups.

Genetic heterogeneity in Leigh syndrome: Highlighting treatable and novel genetic causes.

Leigh syndrome (LS), the most common childhood mitochondrial disorder, has characteristic clinical and neuroradiologic features. Mutations in more than 75 genes have been identified in both the mitochondrial and nuclear genome, implicating a high degree of genetic heterogeneity in LS. To profile these genetic signatures and understand the pathophysiology of LS, we recruited 64 patients from 62 families who were clinically diagnosed with LS at Seoul National University Children's Hospital. Mitochondrial genetic analysis followed by whole-exome sequencing was performed on 61 patients. Pathogenic variants in mitochondrial DNA were identified in 18 families and nuclear DNA mutations in 22. The following 17 genes analyzed in 40 families were found to have genetic complexity: MTATP6, MTND1, MTND3, MTND5, MTND6, MTTK, NDUFS1, NDUFV1, NDUFAF6, SURF1, SLC19A3, ECHS1, PNPT1, IARS2, NARS2, VPS13D, and NAXE. Two treatable cases had biotin-thiamine responsive basal ganglia disease, and another three were identified as having defects in the newly recognized genes (VPS13D or NAXE). Variants in the nuclear genes that encoded mitochondrial aminoacyl tRNA synthetases were present in 27.3% of cases. Our findings expand the genetic and clinical spectrum of LS, showing genetic heterogeneity and highlighting treatable cases and those with novel genetic causes.

Mutational landscape of uterine and ovarian carcinosarcomas implicates histone genes in epithelial-mesenchymal transition.

Carcinosarcomas (CSs) of the uterus and ovary are highly aggressive neoplasms containing both carcinomatous and sarcomatous elements. We analyzed the mutational landscape of 68 uterine and ovarian CSs by whole-exome sequencing. We also performed multiregion whole-exome sequencing comprising two carcinoma and sarcoma samples from six tumors to resolve their evolutionary histories. The results demonstrated that carcinomatous and sarcomatous elements derive from a common precursor having mutations typical of carcinomas. In addition to mutations in cancer genes previously identified in uterine and ovarian carcinomas such as TP53, PIK3CA, PPP2R1A, KRAS, PTEN, CHD4, and BCOR, we found an excess of mutations in genes encoding histone H2A and H2B, as well as significant amplification of the segment of chromosome 6p harboring the histone gene cluster containing these genes. We also found frequent deletions of the genes TP53 and MBD3 (a member with CHD4 of the nucleosome remodeling deacetylase complex) and frequent amplification of chromosome segments containing the genes PIK3CA, TERT, and MYC Stable transgenic expression of H2A and H2B in a uterine serous carcinoma cell line demonstrated that mutant, but not wild-type, histones increased expression of markers of epithelial-mesenchymal transition (EMT) as well as tumor migratory and invasive properties, suggesting a role in sarcomatous transformation. Comparison of the phylogenetic relationships of carcinomatous and sarcomatous elements of the same tumors demonstrated separate lineages leading to these two components. These findings define the genetic landscape of CSs and suggest therapeutic targets for these highly aggressive neoplasms.

DNA-Binding Properties of New Fluorescent AzaHx Amides: Methoxypyridylazabenzimidazolepyrroleimidazole/pyrrole.

DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.

Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA.

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis.

Uncontrolled diabetes as a potential risk factor in tibiotalocalcaneal fusion using a retrograde intramedullary nail.

BACKGROUND: Tibiotalocalcaneal (TTC) fusion using a retrograde intramedullary (IM) nail is an effective salvage option for terminal-stage hindfoot problems. However, as many patients who receive TTC fusion bear unfavorable medical comorbidities, the risk of nonunion, infection and other complications increases. This study was performed to identify the factors influencing outcomes after TTC fusion using a retrograde IM nail. METHODS: Between September 2008 and February 2012, 34 consecutive patients received TTC fusion using a retrograde IM nail for limb salvage. All patients had a minimum follow-up of two years. Throughout follow-up, standard ankle radiography was performed along with clinical outcome assessment using a visual analog scale (VAS) for pain, the American Orthopaedic Foot and Ankle Society Ankle-Hind Foot Scale (AOFAS A/H scale) and the Foot and Ankle Outcome Score (FAOS). For the retrospective analysis, demographic factors, preoperative medical status, laboratory markers, and etiology were comprehensively reviewed using medical records. The success of the index operation was determined using clinical and radiological outcomes. Finally, the effect of each factor on failure after the operation was analyzed using univariate logistic regression. RESULTS: In a mean of seven months, 82% (28/34) achieved union, as evaluated by standard radiography. All clinical outcome parameters improved significantly after the operation, including VAS, AOFAS A/H scale, and FAOS (P<0.001). At the last follow-up, five cases of nonunion with less than AOFAS A/H scale of 80 and two cases of below knee amputation due to uncontrolled infection were determined to be failures. None of the factors (etiology, demographics, laboratory markers and medical status) significantly influenced failures. However, uncontrolled DM significantly increased the failure rate with an odds ratio of 10 (P=0.029). CONCLUSIONS: TTC fusion with a retrograde intramedullary nail is a successful treatment for complicated hindfoot problems such as traumatic osteoarthritis, Charcot arthropathy and failed TAA. However, it should be used judiciously in patients with uncontrolled DM, as the risk of failure increases. DESIGN: Retrospective cohort study.

Hyaluronic acid dressing (Healoderm) in the treatment of diabetic foot ulcer: A prospective, randomized, placebo-controlled, single-center study.

Fast and complete healing of a diabetic foot ulcer (DFU) is challenging due to the hostile wound healing environment of the diabetic patients. As a part of a multimodal treatment approach, advanced dressing material using hyaluronic acid (HA) has been found to be effective. However, previous studies have used HA with additional biologics, which interferes in determining the true clinical effect of HA in DFU. To examine the sole effectiveness of HA in DFU treatment, a prospective, randomized, placebo-controlled, single-center study was conducted using an HA dressing without additional substances. Thus, 34 patients who met the inclusion criteria were randomized into two groups (the study group: HA dressing material; the control group: conventional dressing material). During the 12-week study period, complete ulcer healing rate was evaluated as a primary endpoint. Additionally, healing velocity and the mean duration for achieving a 50% ulcer size reduction was compared between the two groups as a secondary endpoint. At the end of the study, the study group presented a significantly higher complete healing rate as compared to that in the control group [84.6% (11/13), 41.6% (5/12), respectively, P = 0.041]. Additionally, faster ulcer healing velocity and shorter mean duration for achieving a 50% ulcer size reduction were observed in the study group (P = 0.022 and 0.004, respectively). The Kaplan-Meier survival analysis for the median time for 50% ulcer healing rate also showed a significantly shorter duration in the study group (21 days vs. 39 days, P = 0.0127). Finally, there were no adverse events related to the dressing materials used in the study. As a major component of the extracellular matrix, this study supports the safety and efficacy of a pure HA dressing without additional substances in treating DFU.

Minimally Invasive Calcaneal Displacement Osteotomy Site Using a Reference Kirschner Wire: A Technique Tip.

As a standard open approach, the lateral oblique incision has been widely used for calcaneal displacement osteotomy. However, just as with other orthopedic procedures that use an open approach, complications, including wound healing problems and neurovascular injury in the heel, have been reported. To help avoid these limitations, a percutaneous technique using a Shannon burr for calcaneal displacement osteotomy was introduced. However, relying on a free-hand technique without direct visualization at the osteotomy site has been a major obstacle for this technique. To address this problem, we developed a technical tip using a reference Kirschner wire. A reference Kirschner wire technique provides a reliable and accurate guide for minimally invasive calcaneal displacement osteotomy. Also, the technique should be easy to learn for surgeons new to the procedure.

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

SOX2 is crucial for the maintenance of the self-renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)-derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self-renewal and differentiation capacities of BM-MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM-MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM-MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA-mediated acetylation and ubiquitination of SOX2 in BM-MSCs. SIRT1 activation by resveratrol enhanced the colony-forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose-dependent manner. However, the enhancement of self-renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1-SOX2 axis plays an important role in maintaining the self-renewal capability and multipotency of BM-MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post-translational modification in BM-MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1-mediated deacetylation contributes to maintaining SOX2 protein in the nucleus.

AzaHx, a novel fluorescent, DNA minor groove and G·C recognition element: Synthesis and DNA binding properties of a p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (azaHx-PI) polyamide.

The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)).

Inhibition of human glutathione transferases by dinitronaphthalene derivatives.

Glutathione transferase (GST) enzymes catalyze the conjugation of glutathione with reactive functional groups of endogenous compounds and xenobiotics, including halonitroaromatics. 1-Chloro-2,4-dinitrobenzene (CDNB) is one of the most commonly used substrates for GST activity assays. We have studied the interactions of dinitronaphthalene analogues of CDNB with recombinant human GST enzymes (Alpha, Mu, and Pi classes) expressed in Escherichia coli. Dinitronaphthalene derivatives were found to be GST inhibitors. The highest potency of inhibition was observed towards Mu-class GSTs, M1-1 and M2-2; IC50 values for 1-methoxy- and 1-ethoxy-2,4-dinitronaphthalene were in the high nanomolar to low micromolar range. Inhibition accompanies the formation, at the enzyme active site, of very stable Meisenheimer complex intermediates.

High-output heart failure due to a giant left circumflex coronary aneurysm with a left atrial fistula and atrial septal defect: a case report.

Background: A coronary artery aneurysm is a rare cardiac anomaly that may be incidentally detected on echocardiography. When associated with a coronary cameral fistula, an aneurysm can become symptomatic. We present a unique case of a giant left circumflex coronary aneurysm with a fistula to the left atrium and a large atrial septal defect causing acute heart failure in a young woman during the peripartum period. Case summary: A 32 year-old woman who presented with hypoxia after the delivery of her fourth child was found to have heart failure with severe mitral regurgitation and multiple abnormal intracardiac shunts. Echocardiography showed a large circular structure with Doppler color flow into the left atrium and between the atria. Cardiac computed tomography showed multiple dilated coronary arteries including a left circumflex coronary artery aneurysm measuring >10 cm in diameter with fistulous communication to the left atrium and a large atrial septal defect. A right heart catheterization was performed, and the patient was diagnosed with high-output heart failure. Surgical closure of the coronary cameral fistula was deferred due to the risk of worsening pressure in the coronary aneurysm, and the patient was referred for cardiac transplantation. Discussion: This case illustrates severe heart failure as a complication of a giant coronary artery aneurysm with fistulization to the left atrium and subsequent shunting through a large atrial defect. Echocardiography allows for the detection of a coronary aneurysm and shunting, and cardiac computed tomography provides detailed visualization of a coronary cameral fistula.

Conformational modulation of DNA by polyamide binding: structural effects of f-Im-Py-Im based derivatives on 5'-ACGCGT-3'.

The DNA sequence 5'-ACGCGT-3' is in the core site of the Mlu 1 cell-cycle box, a transcriptional element in the promoter region of human Dbf4 gene that is highly correlated with a large number of aggressive solid cancers. The polyamide formamido-imidazole-pyrrole-imidazole-amine(+) (f-Im-Py-Im-Am(+) ) can target the minor groove of 5'-ACGCGT-3' as an antiparallel stacked dimer and has shown good activity in inhibiting transcription factor binding. Recently, f-Im-Py-Im-Am(+) derivatives that involve different orthogonally positioned substituents were synthesized to target the same binding site, and some of them have displayed improved binding and pharmacological properties. In this study, the gel electrophoresis-ligation ladders assay was used to evaluate the conformational effects of f-Im-Py-Im-Am(+) and derivatives on the target DNA, an essential factor for establishing the molecular basis of polyamide-DNA complexes and their transcription factor inhibition. The results show that the ACGCGT site in DNA has a relatively wide minor groove and a B-form like overall structure. After binding with f-Im-Py-Im-Am(+) derivatives, the DNA conformation is changed as indicated by the different mobilities in the gel. These conformational effects on DNA will at least help to point to the mechanism for the observed Mlu 1 inhibition activity of these polyamides. Therefore, modulating DNA transcription by locking the DNA shape or altering the minor groove geometry to affect the binding affinity of certain transcription factors is an attractive possible therapeutic mechanism for polyamides. Some of the substituents are charged with electrostatic interactions with DNA phosphate groups, and their charge effects on DNA gel mobility have been observed.

Affinity and kinetic modulation of polyamide-DNA interactions by N-modification of the heterocycles.

Synthetic N-methyl imidazole and N-pyrrole containing polyamides (PAs) that can form "stacked" dimers can be programmed to target and bind to specific DNA sequences and control gene expression. To accomplish this goal, the development of PAs with lower molecular mass which allows for the molecules to rapidly penetrate cells and localize in the nucleus, along with increased water solubility, while maintaining DNA binding sequence specificity and high binding affinity is key. To meet these challenges, six novel f-ImPy*Im PA derivatives that contain different orthogonally positioned moieties were designed to target 5'-ACGCGT-3'. The synthesis and biophysical characterization of six f-ImPy*Im were determined by CD, ΔTM, DNase I footprinting, SPR, and ITC studies, and were compared with those of their parent compound, f-ImPyIm. The results gave evidence for the minor groove binding and selectivity of PAs 1 and 6 for the cognate sequence 5'-ACGCGT-3', and with strong affinity, Keq = 2.8 × 10(8) M(-1) and Keq = 6.2 × 10(7) M(-1), respectively. The six novel PAs presented in this study demonstrated increased water solubility, while maintaining low molecular mass, sequence specificity, and binding affinity, addressing key issues in therapeutic development.

Hx-amides: DNA sequence recognition by the fluorescent Hx (p-anisylbenzimidazole)•pyrrole and Hx•imidazole pairings.

Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.

Design, synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino, N-formamido, pyrrole- and imidazole-containing polyamides.

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K(eq)=2.4×10(8) M(-1)) and with comparable sequence selectivity to its cognate sequence 5'-ACGCGT-3' when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5'-ACGCGT-3' via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5'-ATGCAT-3' (K(eq)=7.4×10(6) M(-1)) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5'-AAATTT-3' (K(eq)=4.8×10(7) M(-1)), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5'-ATCGAT-3' as a stacked dimer and it has the lowest affinity among the diamino PAs tested (Keq <1×10(5) M(-1)). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the 'core rules' of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.