Accelerated RNA secondary structure design using preselected sequences for helices and loops.

Nucleic acids can be designed to be nano-machines, pharmaceuticals, or probes. RNA secondary structures can form the basis of self-assembling nanostructures. There are only four natural RNA bases, therefore it can be difficult to design sequences that fold to a single, specified structure because many other structures are often possible for a given sequence. One approach taken by state-of-the-art sequence design methods is to select sequences that fold to the specified structure using stochastic, iterative refinement. The goal of this work is to accelerate design. Many existing iterative methods select and refine sequences one base pair and one unpaired nucleotide at a time. Here, the hypothesis that sequences can be preselected in order to accelerate design was tested. To this aim, a database was built of helix sequences that demonstrate thermodynamic features found in natural sequences and that also have little tendency to cross-hybridize. Additionally, a database was assembled of RNA loop sequences with low helix-formation propensity and little tendency to cross-hybridize with either the helices or other loops. These databases of preselected sequences accelerate the selection of sequences that fold with minimal ensemble defect by replacing some of the trial and error of current refinement approaches. When using the database of preselected sequences as compared to randomly chosen sequences, sequences for natural structures are designed 36 times faster, and random structures are designed six times faster. The sequences selected with the aid of the database have similar ensemble defect as those sequences selected at random. The sequence database is part of RNAstructure package at http://rna.urmc.rochester.edu/RNAstructure.html.

Host-driven temperature dependence of Deformed wing virus infection in honey bee pupae.

The temperature dependence of infection reflects changes in performance of parasites and hosts. High temperatures often mitigate infection by favoring heat-tolerant hosts over heat-sensitive parasites. Honey bees exhibit endothermic thermoregulation-rare among insects-that can favor resistance to parasites. However, viruses are heavily host-dependent, suggesting that viral infection could be supported-not threatened-by optimum host function. To understand how temperature-driven changes in performance of viruses and hosts shape infection, we compared the temperature dependence of isolated viral enzyme activity, three honey bee traits, and infection of honey bee pupae. Viral enzyme activity varied <2-fold over a > 30 °C interval spanning temperatures typical of ectothermic insects and honey bees. In contrast, honey bee performance peaked at high (≥ 35 °C) temperatures and was highly temperature-sensitive. Although these results suggested that increasing temperature would favor hosts over viruses, the temperature dependence of pupal infection matched that of pupal development, falling only near pupae's upper thermal limits. Our results reflect the host-dependent nature of viruses, suggesting that infection is accelerated-not curtailed-by optimum host function, contradicting predictions based on relative performance of parasites and hosts, and suggesting tradeoffs between infection resistance and host survival that limit the viability of bee 'fever'.

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-ß-d-2'-deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6-amino-3-(1'-ß-d-2'-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, abbreviated as P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find G-Z pairs have stability comparable to that of A-T pairs and should therefore be included as base pairs in structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include the P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing it with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases in which Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

DNA Structure Design Is Improved Using an Artificially Expanded Alphabet of Base Pairs Including Loop and Mismatch Thermodynamic Parameters.

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo-[1,2- a ]-1,3,5-triazin-(8 H )-4-one and 6-amino-3-(1'-ß-D-2'-deoxyribofuranosyl)-5-nitro-(1 H )-pyridin-2-one, simply P and Z. To obtain the thermodynamic parameters needed to include P-Z pairs in the designs, we performed 47 optical melting experiments and combined the results with previous work to fit a new set of free energy and enthalpy nearest neighbor folding parameters for P-Z pairs and G-Z wobble pairs. We find that G-Z pairs have stability comparable to A-T pairs and therefore should be considered quantitatively by structure prediction and design algorithms. Additionally, we extrapolated the set of loop, terminal mismatch, and dangling end parameters to include P and Z nucleotides. These parameters were incorporated into the RNAstructure software package for secondary structure prediction and analysis. Using the RNAstructure Design program, we solved 99 of the 100 design problems posed by Eterna using the ACGT alphabet or supplementing with P-Z pairs. Extending the alphabet reduced the propensity of sequences to fold into off-target structures, as evaluated by the normalized ensemble defect (NED). The NED values were improved relative to those from the Eterna example solutions in 91 of 99 cases where Eterna-player solutions were provided. P-Z-containing designs had average NED values of 0.040, significantly below the 0.074 of standard-DNA-only designs, and inclusion of the P-Z pairs decreased the time needed to converge on a design. This work provides a sample pipeline for inclusion of any expanded alphabet nucleotides into prediction and design workflows.

Can simulation replace part of clinical time? Two parallel randomised controlled trials.

CONTEXT: Education in simulated learning environments (SLEs) has grown rapidly across health care professions, yet no substantive randomised controlled trial (RCT) has investigated whether SLEs can, in part, substitute for traditional clinical education. METHODS: Participants were physiotherapy students (RCT 1, n = 192; RCT 2, n = 178) from six Australian universities undertaking clinical education in an ambulatory care setting with patients with musculoskeletal disorders. A simulated learning programme was developed as a replica for clinical education in musculoskeletal practice to replace 1 week of a 4-week clinical education placement. Two SLE models were designed. Model 1 provided 1 week in the SLE, followed by 3 weeks in clinical immersion; Model 2 offered training in the SLE in parallel with clinical immersion during the first 2 weeks of the 4-week placement. Two single-blind, multicentre RCTs (RCT 1, Model 1; RCT 2, Model 2) were conducted using a non-inferiority design to determine if the clinical competencies of students part-educated in SLEs would be any worse than those of students educated fully in traditional clinical immersion. The RCTs were conducted simultaneously, but independently. Within each RCT, students were stratified on academic score and randomised to either the SLE group or the control ('Traditional') group, which undertook 4 weeks of traditional clinical immersion. The primary outcome measure was a blinded assessment of student competency conducted over two clinical examinations at week 4 using the Assessment of Physiotherapy Practice (APP) tool. RESULTS: Students' achievement of clinical competencies was no worse in the SLE groups than in the Traditional groups in either RCT (Margin [Δ] ≥ 0.4 difference on APP score; RCT 1: 95% CI - 0.07 to 0.17; RCT 2: 95% CI - 0.11 to 0.16). CONCLUSIONS: These RCTs provide evidence that clinical education in an SLE can in part (25%) replace clinical time with real patients without compromising students' attainment of the professional competencies required to practise.

Student approaches for learning in medicine: what does it tell us about the informal curriculum?

BACKGROUND: It has long been acknowledged that medical students frequently focus their learning on that which will enable them to pass examinations, and that they use a range of study approaches and resources in preparing for their examinations. A recent qualitative study identified that in addition to the formal curriculum, students are using a range of resources and study strategies which could be attributed to the informal curriculum. What is not clearly established is the extent to which these informal learning resources and strategies are utilized by medical students. The aim of this study was to establish the extent to which students in a graduate-entry medical program use various learning approaches to assist their learning and preparation for examinations, apart from those resources offered as part of the formal curriculum. METHODS: A validated survey instrument was administered to 522 medical students. Factor analysis and internal consistence, descriptive analysis and comparisons with demographic variables were completed. The factor analysis identified eight scales with acceptable levels of internal consistency with an alpha coefficient between 0.72 and 0.96. RESULTS: Nearly 80% of the students reported that they were overwhelmed by the amount of work that was perceived necessary to complete the formal curriculum, with 74.3% believing that the informal learning approaches helped them pass the examinations. 61.3% believed that they prepared them to be good doctors. A variety of informal learning activities utilized by students included using past student notes (85.8%) and PBL tutor guides (62.7%), and being part of self-organised study groups (62.6%), and peer-led tutorials (60.2%). Almost all students accessed the formal school resources for at least 10% of their study time. Students in the first year of the program were more likely to rely on the formal curriculum resources compared to those of Year 2 (p = 0.008). CONCLUSIONS: Curriculum planners should examine the level of use of informal learning activities in their schools, and investigate whether this is to enhance student progress, a result of perceived weakness in the delivery and effectiveness of formal resources, or to overcome anxiety about the volume of work expected by medical programs.

Four decades of complaints to a State Medical Board about graduates from one medical school: implications for change in self-regulation processes.

In the context of the impending national registration of health practitioners in Australia including doctors, this column describes the types and patterns of complaints to a State Medical Board across an extended period, about graduates from one medical school. De-identified data concerning complaints about medical practitioners who had graduated from the University of Queensland, made to the Medical Board of Queensland between 1968 and 2006, were analysed. The main outcome measures were category of complaint, total complaint rate per doctor-year, frequency of complaints per practitioner and outcomes of complaints. There were 12 categories of complaints, encompassing different aspects of clinical management, impairment and unethical conduct. Outcomes included "no further action", a hierarchy of recommendations and conditions on registration, suspension, deregistration, health assessment, or referral to alternative bodies. Complaints predominantly related to clinical standards, and this also applied to those who attracted multiple complaints. Most cases were managed without resort to sanctions of any kind. Sanctions may be underutilised, particularly in cases of apparent recalcitrance. Improved tracking and appropriate reeducation and disciplinary measures will assist in better protecting the public under the new national registration arrangements.

Natural Product Medicines for Honey Bees: Perspective and Protocols.

The western honey bee remains the most important pollinator for agricultural crops. Disease and stressors threaten honey bee populations and productivity during winter- and summertime, creating costs for beekeepers and negative impacts on agriculture. To combat diseases and improve overall bee health, researchers are constantly developing honey bee medicines using the tools of microbiology, molecular biology and chemistry. Below, we present a manifesto alongside standardized protocols that outline the development and a systematic approach to test natural products as 'bee medicines.' These will be accomplished in both artificial rearing conditions and in colonies situated in the field. Output will be scored by gene expression data of host immunity, bee survivorship, reduction in pathogen titers, and more subjective merits of the compound in question. Natural products, some of which are already encountered by bees in the form of plant resins and nectar compounds, provide promising low-cost candidates for safe prophylaxis or treatment of bee diseases.

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers.

Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5' end (LNA-5'), near the 3' end (LNA-3') and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, C(T)) were characterized using two-way ANOVA. LNA-5' increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5' generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3' and LNA-Even did not improve read lengths or C(T). ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence.

"Experiments of Nature" and the New Era of Immunology: an historical perspective.

This review of the events leading to the New Era of Immunology focuses on the important leads provided by the Experiments of Nature. Not only did they provide the path that led to our present knowledge of the ontogeny of the immune system, but also to an entirely new perspective of lymphocytic malignancies. The power of taking clinical clues to the laboratory to conduct experiments not otherwise envisioned and then returning with that information to impact the clinical problem that motivated the studies is elaborated. Finally, the ongoing utility of this approach is emphasized.

Biophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P).

Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°37) for oligonucleotide hybridization were extracted. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair observed for C:O6-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodynamics of oligonucleotides containing Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.

Lymphocyte alloantigenic challenge as a potential cofactor in HIV infection and progression to AIDS.

A hypothesis is developed that circulatory exposure to alloantigens found on lymphocytes triggers a series of events leading to suppression of the cell-mediated arm of the immune system and, therefore, that such alloantigenic challenge can be an important cofactor promoting infection with the human immunodeficiency virus (HIV) and development of the acquired immunodeficiency syndrome (AIDS) following infection. The explanatory model that is developed is based in large part on current knowledge of the results of fetal lymphocyte alloantigenic challenge of the maternal immune system. It is our contention that the maternal response which leads to suppression of cell-mediated immunity is a mechanism promoting survival of the fetal "allograft," and that this mechanism is an evolutionary adaptation in humans and other placental mammals. We also hypothesize that circulatory exposure to these same alloantigens leads to an immunosuppressive state which enhances survival of HIV in an exposed individual and augments the immunosuppressive action of HIV in promoting progression to clinical AIDS. Findings from studies of individuals who are at risk for exposure to lymphocyte alloantigenic challenge and from HIV infected patients are reviewed and discussed in light of this hypothesis.

Associations between maternal and fetal serum levels of immune activation markers and fetal growth.

During pregnancy, signs of maternal immunologic sensitization to fetal HLA and other fetoplacental alloantigens are often detectable in peripheral blood. Presumbly, this in part reflects immune activity at the maternal-fetal interface. This response may involve activation of maternal T cells, which stimulate placental growth via lymphokine production. To shed light on this mechanism, data on placental weight, neonatal anthropometry, gestational age, fetomaternal HLA relationships (reflecting a potential for HLA allosensitization), and serum levels of three immune activation markers in maternal and cord blood were collected in a sample of 61 primiparous women and their neonates. The activation markers were soluble CD8 antigen (sCD8), interleukin-2 receptor (sIL-2R), and beta-2 microglobulin (ß2 m). Mean fetal and maternal sCD8 and ß2 m levels, and mean fetal sIL-2R levels were significantly higher than published norms. Fetal means for all three markers exceeded maternal means, and both sIL-2R and ß2 m were highly correlated between mother and fetus. This suggests that fetal sIL-2R and ß2 m levels result in part from transport or diffusion from the maternal compartment. No associations were found between fetomaternal HLA relationships, activation markers, and placental weight. The difference between the fetal and maternal ß2 m value was significantly correlated with birth weight, controlling for chest circumference and crown-heel length. Associations between birth weight and fetomaternal HLA relationships could not be interpreted with certainty. These findings suggest that maternal immune activation and diffusion or transport of ß2 m into fetal compartment enhances fetal growth in fat-free body mass. © 1995 Wiley-Liss, Inc.

Assessment of methadone clinic staff attitudes toward hepatitis C evaluation and treatment.

We used a 25-item, self-administered questionnaire to assess staff's perceived barriers and willingness to engage in onsite treatment of hepatitis C virus (HCV) at the Beth Israel Medical Center methadone maintenance treatment program (MMTP) at its Harlem sites. Of 80 participants, 50% were counselors and 24% were directly involved in referral or HCV testing. Although 92% of the MMTP staff indicated that they discuss HCV evaluation and treatment with patients at least annually, 70% believed that less than 25% of patients accept referral for HCV treatment and attend their initial appointment. Most staff (66%) supported onsite HCV evaluation and treatment, although support was higher among those with a bachelor's degree or higher (p = 0.046). Lack of infrastructure was perceived as the greatest obstacle to onsite treatment. Educational interventions and skill building for staff to confidently engage and support MMTP patients in HCV treatment may be necessary prerequisites for onsite HCV management in MMTPs.

Simulation can contribute a part of cardiorespiratory physiotherapy clinical education: two randomized trials.

INTRODUCTION: Simulated learning environments (SLEs) are used worldwide in health professional education, including physiotherapy, to train certain attributes and skills. To date, no randomized controlled trial (RCT) has evaluated whether education in SLEs can partly replace time in the clinical environment for physiotherapy cardiorespiratory practice. METHODS: Two independent single-blind multi-institutional RCTs were conducted in parallel using a noninferiority design. Participants were volunteer physiotherapy students (RCT 1, n = 176; RCT 2, n = 173) entering acute care cardiorespiratory physiotherapy clinical placements. Two SLE models were investigated as follows: RCT 1, 1 week in SLE before 3 weeks of clinical immersion; RCT 2, 2 weeks of interspersed SLE/clinical immersion (equivalent to 1 SLE week) within the 4-week clinical placement. Students in each RCT were stratified on academic grade and randomly allocated to an SLE plus clinical immersion or clinical immersion control group. The primary outcome was competency to practice measured in 2 clinical examinations using the Assessment of Physiotherapy Practice. Secondary outcomes were student perception of experience and clinical educator and patient rating of student performance. RESULTS: There were no significant differences in student competency between the SLE and control groups in either RCT, although students in the interspersed group (RCT 2) achieved a higher score in 5 of 7 Assessment of Physiotherapy Practice standards (all P < 0.05). Students rated the SLE experience positively. Clinical educators and patients reported comparability between groups. CONCLUSIONS: An SLE can replace clinical time in cardiorespiratory physiotherapy practice. Part education in the SLE satisfied clinical competency requirements, and all stakeholders were satisfied.

Medical school selection criteria and the prediction of academic performance.

OBJECTIVE: To assess how well prior academic performance, admission tests, and interviews predict academic performance in a graduate medical school. DESIGN, SETTING AND PARTICIPANTS: Analysis of academic performance of 706 students in three consecutive cohorts of the 4-year graduate-entry medical program at the University of Queensland. MAIN OUTCOME MEASURES: Proportion of academic performance during the medical program explained by selection criteria, and correlation between selection criteria and performance. Selection criteria were grade point average (GPA), GAMSAT (Graduate Australian Medical School Admissions Test) score, and interview score. Academic performance was defined as overall total in all examinations combined, in first and fourth year examinations, and in individual written, ethics and clinical components. RESULTS: Selection criteria explained 21.9% of variation in overall total score, falling from 28.2% in Year 1 to 17.7% in Year 4. This was highest for the written examination in Year 1 (30.5%) and lowest for the clinical examination in Year 4 (10.9%). GPA was most strongly correlated with academic performance (eg, for overall score, partial Spearman's correlation coefficient [pSCC], 0.47; P < 0.001), followed by interviews (pSCC, 0.12; P = 0.004) and GAMSAT (pSCC, 0.07; P = 0.08). The association between GPA and performance waned from Year 1 to Year 4, while the association between interview score and performance increased from Year 1 to Year 4. CONCLUSION: The school's selection criteria only modestly predict academic performance. GPA is most strongly associated with performance, followed by interview score and GAMSAT score. The school has changed its selection process as a result.

The "pyramid of professionalism": seven years of experience with an integrated program of teaching, developing, and assessing professionalism among medical students.

The authors report on an integrated program of teaching, developing, and assessing professionalism as well as managing unprofessional behavior referrals and supporting students through the Personal and Professional Development Committee (PPDC) in the four-year, graduate-entry medical program at the School of Medicine, University of Queensland, Australia. Two thousand six hundred thirty medical students have participated in the ethics and professional practice teaching program from 2000 to 2006. They were assessed through formal examination; students who did not satisfy requirements completed supplementary examinations. One student failed a year on the basis of formal examination. Instructors referred 507 students (19% of all enrolled) during the seven-year period to the PPDC, which interviewed 142 (25%; 5% of all enrolled) at least once; 25 of these more than once. In all, 711 reports were submitted to the PPDC, 420 (55%) for unsatisfactory attendance only and 291 (45%) for other concerns. Most of these related to "responsibility/reliability" (46.7%) and "participation" (41.9%);12.4% related to "honesty/integrity [corrected] The PPDC referred four students to the board of examiners, and two students failed a year for persistent unprofessional behavior. The authors established a Pyramid of Professionalism whose foundation is a formal curriculum of medical ethics, law, and professionalism. At higher levels, the pyramid mirrors Australia's medical regulatory processes, combining nonpunitive support with the possibility of sanctions, by mediating and sometimes remediating a range of notified concerns. Students who persist in behaving unprofessionally or in seriously unacceptable ways have failed academically on professionalism grounds.

Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation.

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaH(o), DeltaS(o), and melting temperature (T(M)) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaH(o), DeltaDeltaS(o), DeltaDelta, and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaH(o) and DeltaDeltaS(o) is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaH(o), DeltaDeltaS(o), and DeltaDelta parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MX(L) and 5' X(L)N, where M, N, and X = A, C, G, or T and X(L) represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T(M) estimates accurate to within 2 degrees C for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.